DEPARTMENT OF BOTANY PLANT GENETIC ENGINEERING LABORATORY

THE UNIVERSITY OF QUEENSLAND

Brisbane, Qld 4072, Australia
Telephone (07) 3365 1128
Lab Telephone (07) 3365 4686
FAX (07) 3365 1699

Basic Molecular Biology Techniques

and
Lab Procedures

Edition 10.1

Updated 15/6/2001
 ii

Table of Contents
1. Introduction 1
About this manual 1
2. General Procedures 2
Dilution equations 2
Converting concentrations mg/mL to M (molar): 3
Quantifying DNA using GelWorks 1D Advanced 4
GCG Fragment assembly program 4
Using the spectrophotometer 6
3. DNA Procedures 7
Ligations 7
Transformation of E.coli 7
Modified miniprep method 8
Bacterial cracking preps (Malaga Style) 9
Rapid colony screening 10
PEG purification of plasmid DNA 11
Phenol-SEVAG / ethanol precipitation of DNA 11
Miniprep digestion by restriction endonucleases 11
Extract-O-mutant 12
Genomic DNA processing 13
CsCl purification of genomic DNA 14
Restriction digestion of genomic DNA for Southern analysis 15
4. RNA Procedures 16
RNA tips and tricks 16
Hot Phenol RNA Extraction 17
Arabidopsis RNA extraction procedure 18
The “Pineapple” RNA extraction procedure 19
RNA electrophoresis 20
RNA solutions 21
5. Hybridisation 22
Radiation safety 22
Capillary blotting of Northerns and Southerns 23
Posi-blot transfer of Northerns and Southerns 25
 iii

Northern and Southern hybridisation 27

Striping membranes 29
6. PCR Techniques 30
Reverse transcription polymerase chain reaction 30
Preparation of 10mM dNTP 31
Blunt end cloning of PCR products (And how to make CIAP EcoRV pBS) 32
7. Protein Techniques 34
Fun with PAGE and westerns 34
PAGE and western solutions 37
ELISA 39
ELISA solutions 40
Bradford assay 41
8. Plant tissue culture and transformation 43
Agrobacterium tri-parental mating 43
Arabidopsis vacuum infiltration (in planta) transformation 45
Arabidopsis floral dip transformation 48
Mung bean transformation 50
Papaya transformation protocol 51
Papaya media 54
Tobacco seed sterilisation 57
Tobacco transformation protocol 58
8.1. Transgenic analysis 60
Transgenic phenotype analysis for Arabidopsis 60
GUS assay 65
GUS fluorometric assay 68
9. Other procedures 71
Exonuclease III digests; (Sequencing using deletions) 71
Phage Hints 72
cDNA library screening 73
Electrocompetent cells 76
in situ Hybridisation 77
I Tissue fixing, embedding and sectioning 77
II Making DIG labelled probe 78
III Hybridisation 79
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10. Appendixes 86
Appendix A: Restriction digests buffer tables and recognition sequences 86
Restriction enzyme regonition sites 87
Appendix B: Solutions 89
Appendix C: Antibiotic solutions chart 93
Appendix D: Looking after your Gilson 94
 1

1. Introduction
About this manual
This manual contains most of the molecular biology procedures performed in this
lab. It has been laid out to be simple to follow. Always check this manual for any
information before seeking help from tired and irritable PhD students, research assistants
and post-docs.
As you use this manual, be aware that not all the solutions are in Appendix B, some
are included with some procedures. And also if the solution you are looking for is not
included within the procedure, then look in Appendix B.
Also this manual is a work in progress, and is updated many times throughout the
year. If you feel something should be added or modified, or if you find errors (speelling,
grammar or scientific errors), let Tony know.
 2

2. General Procedures
Dilution equations
- Generally C * V = C’ * V’ Unit Prefix Factor
Where: m 10-3
C’: Concentration of final solution µ 10-6
V’: Volume of final solution n 10-9
C: Concentration of stock solution p 10-12
V: Volume of stock solution f 10-15
Example 1:
Require 10mL of 1M NaCl solution; Have a stock of 5M NaCl
C = 5M; C’ = 1M; V’ = 10mL; V = Unknown
C * V = C’* V’ 5 * V’ = 10 * 1; V’ = 10 / 5; V’ = 2mL
Therefore add 2mL of 5M NaCl to 8mL ddH2O

Example 2:
Have a 10mL solution which requires a final concentration of 1M NaCl; Have a
stock solution of 5M NaCl
Rearrange above equation, where V’ = X+V
Where:
X: Original volume
V: Amount of stock solution to add
Use this equation: (X+V) * C’ = V * C
Therefore:
X = 10mL; C’ = 1M; C = 5M; V = Unknown
(x+V) * C’ = V * C (10+V) * 1 = V * 5 10 + V = 5V
10 = 4V V = 10 / 4 V = 2.5mL
So add 2.5mL of 5M NaCl to the original 10mL solution
 3

Converting concentrations mg/mL to M (molar):

Useful for primer concentrations

To convert from mg/mL to M;

eg.

Concentration of primer; 1.18mg/mL (µg/µL)

Molecular weight; 6348g/mol

1.18 = 6348
x mol 1

1.18 = x mol
6348 1

x mol = 1.86 x 10-4 M = 185µM

To convert from M to mg/mL

eg
Concentration of primer; 200µM
Molecular weight; 5240g/mol

x µg/µL = 5240
2.00 x 10-4 1

x µg/µL = 5240 x 2.00 x 10-4

x µg/µL = 1.05µg/µL

To work out MW of a primer:

Average MW of a nucleotide = 317.4g/mol/base

Therefore the MW of a 20base primer would be:

20 x 317.4 = 6348g/mol

Note for sequencing primers:

You need 6.4pmol primer per reaction
Therefore a 6.4pmol/µL stock is:

6.4pmol/µL = 6.4 x 106 pmol/L = 6.4 x 10-6 mol/L = 6.4µM

You can now make the appropriate dilution

 4

Quantifying DNA using GelWorks 1D Advanced

Transfer tif file to c:\data\gelworks
Open up GelWorks program
File
New Experiment
Open file
Rotate image if needed so lanes run horizontally (View, rotate clockwise)
Create lanes
Draw a box to include all bands
Accept lane
Done
Check Analysis window – if background is sloped;
Background
Rubber band
Manual
Double click at bottom of peaks to adjust slope
Detect bands
Use right mouse button to delete excess peaks
Add peaks with left mouse button
Move peak edges to encompass bands
Quantity calibration (beaker icon)
Click on blue dot of standard bands
Enter in values for calibrated volumes
Use curve to assign all bands
Go to Lane measurements window and read volumes from table

GCG Fragment Assembly

An ANGIS Program to assemble a consensus sequence

For sequence data retrieved from PALM, this data must be uploaded to your directory at
ANGIS.

• Start Netscape and log into your own directory via WebANGIS Web File Manager.
• In the Windows Explorer, navigate to “Sequence on ‘Palm\Data\Users’(K:). Open the
‘sequence only’ file under your name in the dated folder (date corresponds to when
data was uploaded to PALM).
• This will open in Notepad. Select the entire sequence and copy it to the clipboard. In
the Netscape window, click on the File Manager ‘Edit/Create’ button, and paste the
sequence data into the space present in the window which subsequently opens. Trim
off the vector sequences which are present by simple visual recognition of these from a
polylinker map of the plasmid you are using. Put your file into Staden format by
trimming the sequence into lines of exactly 60 nt (you’ll thank me later!). Give your file
a suitable name.
• When all of the separate files containing the fragments for which you wish to obtain a
consensus are thus uploaded, you may now begin sequence exchanging. For later
purposes, put all of the files you intend to use in a single, independent subdirectory.
 5

• In Netscape, go to the ANGIS Home Page (a place of great wonderment and mystery).
Scroll down and click on the ‘Login to 2D ANGIS’ button., and from there the ‘Click here
to start a Telnet session’ link. (Note that this may be just as easily achieved via the
traditional Telnet route).
• After logging in, select 1,1 Unix Command Mode and use standard DOS commands to
change directory (e.g. cd gooblies) to get to the special directory I told you to put all of
your favourite files in earlier! When you’re done, type ‘mainmenu’.
• Go to 3,1 GCG. Specify the Staden sequence format for sequences in the dialog box
which has appeared. Go to 3,7 Sequence Exchange. Select 8 From staden (i.e. to
GCG). Accept the default parameters, Give the name of the file you want converted
(one at a time please!), and the program will automatically write the sequence to a new
file in the WebANGIS directory you are currently working out of. Use the View button in
the Web File Manager to check that this program has done it’s job properly. You should
now rename all of these files, ending them all in .seq. This is important!
• Now for the fun part – using the GCG Fragment Assembly program to create a contig.
In the GCG program menu, go to 1,4 and toggle the sequence format to GCG (press
ENTER). This is essential as the program we will use expects 9and will only accept)
GCG format, and we must tell it that this is what we intend to give it.
• Now go to 2,6 Frag. Assembly. Select 9. Gelstart, and accept the default parameters.
For a new project, give it a suitable name; if you have an existing contig you wish to
add to, etc. then give its name. Gelstart will tell you if it knows of such a project. If it is a
new one, type y at the prompt. The program will ask if you would like any vector
sequences or restriction sites highlighted…I always say no (by just pressing ENTER) to
both questions. Gelstart will tell you what it knows of the project (i.e. 0 fragments in 0
contigs, because you haven’t entered any sequences yet!). Note that Gelstart has
created a new subdirectory in your pwd (present working directory), so don’t be
alarmed to find this in WebFM after clicking on the reload button.
• Return to the menu, go to 5. Gelenter, and type y at the changing default parameters
prompt. Go down to Enter existing files to database and type in: *.seq. This tells the
program that you want it to include all of the files in the pwd ending in .seq to be
entered into the program. Press CONTROL-X to exit this screen.
• Return to the menu, go to 7. Gelmerge, and accept the default parameters. If the
program does not return a single contig, try progressively dropping the word size (= the
number of contiguous bases which are compared as a unit with the other sequences),
and the minimum overlap length (note that the minimum overlap cannot be smaller
than the word size). Leave the fraction of words in the overlap that must match alone –
it may even need to be decreased if you happen to have some serious sequencing
screw-ups in the region of the overlap. Of course, if you had exact matches in the
overlaps, the fraction matching in the overlap is guaranteed to be 1.0 (= 100%).
• Return to the menu, go to 11. Gelview, and accept the default parameters. Save the
output file as its default name. Now go to WebFM and click on the reload button. View
the contig map that was produced by Gelview. Are all of the fragments incorporated
into the contig map? If not, you may need to start again and use a smaller word size
and overlap length in Gelmerge.
• Return to the menu, go to 3. Gelassemble, and accept the default parameters. The
Gelassemble screen will show the consensus sequence below the overlapping
fragments. Use CONTROL-D to switch into the command mode. You may now edit
your sequences to correct for errors or ambiguities in your sequence. Have your
sequence trace printout with you to do this, so you van visually verify which of the
conflicting data sets you wish to believe. For details on the commands available,
 6

consult pages 14-10 to 14-20 of the ANGIS Tutorial Book, the tome of all ANGIS
wisdom. To leave Gelassemble when you are happy with your results, type exit. This
saves the edited contig, and logs you off the program (NEVER use ‘quit’, which does
not save your work!).
• Now you may view the fruits of your electronic labour via WebFM. Your final output
contig should be a nice, clean, GCG-formatted file whose length and sequence are
consistent with the overlapping fragments from which it was constructed.
• Finally, you may opt to get an additional output by returning to the menu, going to 8.
Gelpicture, and accepting the defaults. Enter the name of your consensus sequence
file, and go check the output via WebFM.

Congratulations! You are now the proud owner of your very own consensus sequence!
Enjoy…

Using the Spectrophotometer

Turn on machine – it takes about 5mins to start up
Turn on UV lamp 15min before use
- Press setup
- Press UV lamp
- Press Lamp on
- Wait 15min
From the menu select Scan λ (wavelength)
Press Options
- Select λ min, set to 220
- Select λ max, set to 320
- Select λ step, set to 1 or 2nm
Place in blank into the cuvette holder
Press Baseline
Add sample into the cuvette
Press Start Scan
Press Cursor and move the cursor until it reads 260
Press Print
Select Print Graph

To work out concentration:

Conc=ABS(260nm) x dilution x C
where:
Nucleic Acid C
RNA 40
DNA 50
ssDNA 33
 7

3. DNA Procedures
Ligations
These DNA amounts will give the correct 3:1 insert to vector ratio (ie. 180 fmol
insert : 60 fmol vector). But always use more insert than calculated (2-5x).
Insert Size DNA req’d Vector Size DNA req’d
1 kb 117 ng 3 kb 117 ng
2 kb 234 ng 4 kb 156 ng
3 kb 351 ng 5 kb 195 ng
Add 6 kb 234 ng
Together:
10 kb 390 ng
Vector DNA As in the
Insert DNA Tables 20 kb 780 ng
Ligase Buffer 2µL
Ligase 2µL 27 kb 1050 ng
Water Up to 20µL
15°C / overnight

Ligations with pGEM-T easy:

Add together:
1µL pGEM-T easy vector
1mL 10X Ligation Buffer
2µL Ligase
(-ATP):
2µL 10X Buffer (with no ATP) 15°C / overnight
300µL 1M Tris pH7.8
1µL ATP (50mM)
100µL 1M MgCl2
2µL 50% PEG 4000
100µL 1M DTT
9-12µL insert DNA
500µL ddH2O
volume to 20µL with ddH2O

Transformation of E.coli
Electroporation (Place all items on ice before and during use):
- Place sliding cuvette holder on ice
- Thaw a tube of electrocompetent DH10B Escherichia coli cells
- {OPTIONAL} Heat ligation 65°C/10min, then on ice {OPTIONAL}
- Add 1-5µL of plasmid DNA to the tube of competent cells
- Mix by contents by pipetting up and down gently without causing air bubbles
- Transfer to a electroporation cuvette and place cuvette on ice
- Turn on electoporator and set power supply to 1.8kV
- Place cuvette in sliding cuvette holder
- Have 960µL SOC ready in a pipetter
- Place cuvette in sliding cuvette holder
- Press both pulse buttons until the tone sounds
- Remove cuvette from holder
- Quickly and gently add the SOC and mix well
- CONTINUE TO RECOVERY BELOW
 8

Recovery:
- Shake the tube horizontally for 1 hour in a 37oC shaker
- At the start of the incubation place plates needed in the 37°C incubator (100µg/mL
LB-Amp Plates)
- After 30min: using a glass spreader plate out 100µL of X-gal (20mg/mL in dimethyl
formamide) on the plates, and place back in the incubator.
- After a further 30min plate 200µL of cells, store cells at 4°C and place plates in the
37°C incubator

Heat Shock (An older method rarely used):

- Thaw a tube of competent DH5α Escherichia coli cells
- Add 10µL of plasmid DNA to the tube of competent cells
- Incubate on ice for 20 - 30 minutes
- Heat shock for 90 seconds at 42oC
- Incubate on ice for 1 - 2 minutes
- Add 800µL of SOC
- CONTINUE TO RECOVERY AS ABOVE

Modified miniprep method

- Using a sterile toothpick pick a single colony and place it into a sterile universal tube
containing 2-5mL LB with 50µg/mL Amp
- Grow the culture O/N at 37°C in the shaking incubator
- Mix 200µL of the miniprep culture with 200µL sterile glycerol solution (30%v/v), and
store tubes at -80°C
- Pellet cells
- 5mL culture into a 15mL tube, centrifuge for 10min/4°C/4500rpm in the Jouan
- 2mL culture into eppendorfs, centrifuge 1min/13,000rpm
- Remove the supernatant using the vacuum line
- Resuspend pellet in 100µL Solution 1 and transfer into an eppendorf tube (if not
already in an eppendorf)
- Vortex until fully resuspended
- Add 200µL solution 2, invert 2-3 times to mix
- Add 150µL solution 3, invert 2-3 times to mix
- Centrifuge 5min in a microfuge
- Remove the supernatant and transfer into a new eppendorf tube
- {OPTIONAL} Centrifuge 5min in a microfuge
You get a much cleaner prep this way {OPTIONAL}
- Remove the supernatant and transfer into a new eppendorf tube
- Add 1000µL 100% ethanol, mix well
- Centrifuge 5min in a microfuge
- Remove ethanol and add 200µL of 70% ethanol
- Centrifuge 5min in a microfuge
- Remove ethanol and dry under vacuum for 15min
- Resuspend pellet in 50µL TE or ddH2O
 9

Bacterial cracking preps (Malaga Style) Andrew

Firstly…
First requirement before using this procedure is for you to think about what you want to
see on the final gel. If you are cloning a small fragment into a large vector (say 500bp
into a 6kb vector) forget it, chum, because you will not see the difference. Similarly, if
your vector is bigger than about 10kb, you are wasting your time as this will co-migrate
in the gel with the bacterial genomic DNA.

Secondly…
Inoculate 2mL LB-Amp cultures (or whatever selection you are using) with single
bacterial colonies. Incubate at 37°C with shaking overnight.
Always include a control sample, which is the vector without an insert. If you are doing
blue/white screening, this is a blue colony. If not, use a colony from a plate of bacteria
containing the vector alone (e.g taken from a glycerol stock). This control is important.
Pour a standard 0.8% agarose, 0.5x TBE gel before you start you preps. You will not
need spare lanes for size markers.
At the bench, transfer a 30µL aliquot of each LABELLED overnight culture into a
LABELLED Eppendorf tube. Put the remainder of the cultures in the fridge.
Add 30µL of phenol-SEVAG and 30µL of cracking buffer (see recipe below).
Vortex each tube for approx. 10 secs.
Centrifuge at maximum speed for 3 min.
Load 10-20µL of the supernatant directly onto your agarose gel and electrophorese for
about 30 min (depending on required degree of band separation). NOTE: Be careful
during loading not to take some of the organic phase with your sample. Your sample
will mysteriously float back out of the well if you do so.

Thirdly…
Interpreting your results: The upper and two lower of the four bands you see are a
constant size in your control and sample lanes. This is the bacterial chromosomal DNA
(upper) and bacterial RNA (lower two). Your plasmid should be running between these.
Recombinant plasmids will show a band shift upwards with respect to your control
vector.
Now, you can miniprep those cultures which correspond to your observed positives. If
you want to sequence these directly, you can add 3mL media to the existing culture
and return it to the shaker for a couple of hours, and then use the Qiagen spin
columns. Finally, if you will be using a lot of this construct, you can use the miniprep
culture as a preculture for a maxiprep.
 10

e.g. Crackings of colonies from a blunt ligation of a 0.9kb and a 1.1kb PCR product into
pBS-EcoRV. Lane 1 is a blue colony as the control. Lanes 2-11 are white colonies from
the 0.9kb insert ligation, and Lanes 12-20 are white colonies from the 1.1kb insert ligation.

Contro 0.9kb ligation 1.1kb ligation

l

Geno

RNA

Rapid colony PCR screening technique Andrew

This procedure is good for screening colonies recovered from a ligation which contain an
insert, by using primers flanking the multiple cloning site. This is a very rapid, one-day
method for screening in plasmids too large for cracking prep analysis.

1. The PCR premix contains:

Component 1x 100x
10x Qiagen PCR Buffer 2 µL 200 µL
25mM MgCl2 1.2 µL 120 µL
10mM dNTPs 0.4 µL 40 µL
15µM Primer #1 0.4 µL 40 µL
15µM Primer #2 0.4 µL 40 µL
H2O 11.6 µL 1160 µL

Component 1x
H2O 3.6 µL
Home-made Taq 0.4 µL
polymerase

Make this premix in an Eppendorf tube. Total reaction volume is 20µL.

2. For each colony to be screened, set up a labelled 0.5mL Eppendorf tube (not a thin-
walled PCR tube) with 3.6µL H2O. Using a yellow tip, touch the labelled bacterial
colony to pick up some cells, and resuspend in the H2O. Repeat (with a fresh tip) for all
colonies to be screened.

3. To screen 10 colonies make a PCR mix of 11x by adding 176µL (=11x16µL) of premix
and 4.4µL (=11x0.4µL) of home-made Taq. Add 16.4µL of this PCR mix to each tube.
 11

4. PCR conditions are:

Temperature (°C) Time

94 1min

94 30sec
35 cycles
72 Xmin
72 15min
4 HOLD

Where x is the length of the expected product in kb.

5. Run out your PCR products on a 0.8% agarose gel. High MW smearing suggests too
much template – pick less cells for each tube.

PEG purification of plasmid DNA

- Add 50µL ddH2O and 1µL RNase (10mg/mL) to miniprep DNA
- Incubate 37°C for 30min
- Add 0.5vol SEVAG and 0.5vol Phenol, vortex and centrifuge for 3min, Remove
upper layer into a new tube repeat above step
- Remove upper layer into a new tube and add 1 vol PEG solution
- Flick tube several times, Contents should turn clear
- Incubate at room temperature for 10min
- Centrifuge for 30min
- Wash pellet with 70% ethanol twice, dry in the desiccator
- Resuspend in 40µL ddH2O
N.B: This procedure does not work well for plasmids over 10Kb

Phenol-SEVAG / ethanol precipitation of DNA

- Add 0.5 vol SEVAG and 0.5 vol Phenol, vortex and centrifuge for 3min, Remove
upper layer into a new tube repeat above step
- Add 0.1 vol 3M sodium acetate (pH5.2), mix, then add 2 vol 100% ethanol
- Incubate at -80°C/30min or -20°C/overnight (Optional)
- Centrifuge for 30min/4°C
- Wash pellet with 70% ethanol, dry in the desiccator
- Resuspend in 50µL TE (pH 8.0)

Miniprep digestion by restriction endonucleases

For 1 digest
- Add the following together:
2µL DNA
1µL RNase (0.1µg/µL)
0.5µL of each enzyme (usually 5u/µL)
Restriction digest buffer (depends on the enzyme, see below)
- Add ddH2O to make final volume up to 10µL
 12

For more than 1 digest

- If more than one sample is being digested make a master mix consisting of
RNase, buffer, enzyme and ddH2O
- For a master mix work out how much enzyme, buffer, etc each sample will need
and multiply by 1 more than the number of samples (eg if you have 10 samples,
multiply by 11)
- Make the master mix and aliquot out into the corresponding tubes, then add the
DNA
- Incubate at 37°C for 2hr N.B SmaI is incubated at room temperature
Restriction Digests Buffers (See Appendix A)

WHEN YOU REMOVE AN ENZYME FROM THE FREEZER MAKE

SURE THAT YOU PLACE IT IN THE COOLER. DO NOT LEAVE
ENZYMES AT ROOM TEMPERATURE OR ON ICE
Yes it’s…
Extract-O-Mutant
What can Extract-O-Mutant do for me?: Extract-o-mutant gives PCR-quality gDNA using
small amounts of tissue for reliable and repeatable results. There is the option of an RNA-
free prep as well. Works with Arabidopsis and tobacco and probably your budgie as well.
Also a great way to combine power tools and molecular biology.

Extract-o-mutant was actually invented by: Patrick Krysan, see PNAS 93(15): 8145-
8150 (1996)

You’ll need to make:

Η Extract-O-Mutant buffer 250ml

0.2M Tris 6.06g or 50ml@1M

0.4M LiCl 4.24g or12.5ml @8M
25mM EDTA 12.5ml @0.5M
1% SDS 25ml @10%

adjust to pH 9.0 Careful - LiCl is toxic

Η Plus
- autoclaved plastic grinder bits
- phenol/sevag prewarmed to RT
- isopropanol (as distinct from iso-amy alcohol- this does not work well)
- drill on a stand
- 1.5mL tubes to grind in. Real ‘Eppendorf’ tubes or similar quality tubes are best because
the normal QSP ones will leak phenol 50% of the time (no matter what some other
people in the lab say)
 13

Here’s how

Η Snap off a small piece of healthy leaf into a microfuge tube with the cap – something
about the size of a couple of day old Arabidopsis cotyledon is fine. You can do this in
the growing room/glasshouse, but keep the tube on ice until you extract it.

Η Add 500µL of buffer to the leaf bit and grind it well. About 20 seconds of grinding
should be fine.

Η Add 500µL of phenol/sevag and vortex for 1 min. If you are doing a lot of samples,
vortex each sample for 30 sec after adding the phenol/sevag and repeat this with all
samples when you have finished grinding and are ready to go on to precipitation.

Η Spin for 5min @13K RT, then remove 400µL of the aqueous phase to a new tube.
This step will remove most, but not all, of the RNA (precipitated by the LiCl)

Η Add 400µL of isopropanol, vortex and spin for 15min @13K RT.

Η Remove as much isopropanol as possible and air dry DNA pellet for 5min on the
bench. No need for a 70% ethanol wash.

Η Dissolve the pellet in 300µL of TE.

Η Use 1.5µL for a PCR reaction, and get ready for fantastic results. The gDNA will shear
a bit with each thawing, so it is a good idea to make aliquots if you think you might be
reusing it many times.

Η If you want an RNA-free prep, redissolve the pellet from step in 400uL of TE, add
RNaseA to 40ug/mL, incubate at 37C for 1hr or so, and precipitate with isopropanol as
before.

Genomic DNA processing Dave

Use this procedure to clean up genomic DNA obtained from an RNA extraction.

Add 2vol ethanol + 0.1 vol 3M NaAc to supernatant from LiCl precipitation and store at
-20°C until you are ready to continue.
Spin in 50 falcon tubes at 4500rpm for 30min at 4°C
Wash twice in 70% ethanol wash and air dry
Resuspend in 400µL water (Do not vortex), pass into 1.5mL tube
Add 50µL RNase(10mg/mL), incubate for 2hrs at 37°C
Extract with phenol/SEVAG extract
To the aqueous layer add 2 vol of ethanol
Centrifuge in a microcentrifuge for 15min at 4°C
 14

Wash the pellet twice in 70% ethanol wash

Air dry, DO NOT VACUUM DRY
Re-suspend in 400µL TE. Store at -20°C, Do not vortex, to thaw out

CsCl purification of genomic DNA Chris

1. Extract genomic DNA using a method of choice and resuspend in TE(4mL-small , 8mL-
medium and 18mL-large amounts of DNA) - use either 15 or 50mL polypropylene tubes.

2. Add an equal amount(g) of solid CsCl and mix by gentle inversion until dissolved. note:
allow for the addition of EtBr( 0.4mL-small, 0.6mL-medium and 1mL-large amounts of
DNA.

3. Add EtBr (7-10mg/mL) and mix gently. note: if the DNA sample contains large amounts
of protein and/or carbohydrates a precipitate may appear- spin (5000rpm for 5min) or
filter through miracloth to remove. WEAR GLOVES AND TAKE ALL PRECAUTIONS
TO PROTECT YOUSELF AND OTHERS. IF IT IS NOT ON, IT’S JUST NOT ON!!!!

4. Transfer to an ultracentrifuge tube and top up the tube with 1g CsCl for every 1mL of
TE. Prepare a balance tube with CsCl-TE and balance the two tubes to the nearest
milligram( close is not good enough!)

5. Ultracentrifuge at 50 000rpm around 15-20oC using the Sorvall T-875 rotor. Ask for
assistance on how to use the ultracentrifuge and rotor!

6. After approx 24 hours stop the ultracentrifuge and allow 30 minutes for the rotor to
stop spinning. During this time set up all the necessary materials to extract the band of
DNA using a needle and syringe.

7. Extract the DNA band using an 18G needle and 3-5mL syringe. For first timers seek
assistance as this is a critical and dangerous step. NO FEAR! Note: Be careful not to
disturb the DNA band. When inserting the needle, gently twist it, so as to prevent
blocking the needle. Pump the syringe before using it.

8. Remove the EtBr with n-butanol extractions until the pink colour of the EtBr has been
removed and then do two more. (n-butanol consists of 1vol butanol and 1 vol water).
note: dispose of EtBr waste into bottle in fume hood.

9. Dilute 3-4 fold in TE and add 2 vol of EtOH at RT or ice for 30 min. DNA should
precipitate immediately as a flocculant mass making it easy to remove without
centrifugation. Centrifuge 5000rpm*30 min and either wash in 70% EtOH(15-50mL) O/N
at 4oC or perform dialysis. note: use nuclease free water

10.Centrifuge as above and reprecipitate with ethanol. Resuspend your DNA in TE and
quantify when ready.

REMEMBER PRACTICE MAKES PERFECT!!!!

 15

Restriction digestion of genomic DNA for Southern

analysis
- For each enzyme set up the following reaction

10X restriction enzyme buffer 30µL

0.1 M spermidine 7.5µL
restriction enzyme 2.5 U/µg DNA
10µg DNA XµL
dH2O up to 300µL

- Incubate at 37°C for at least 3-4 hours (overnight is fine).

- Add 30µl (1/10 V) of 3M NaOAc (pH 5.2) and 750µl (2.5 V) of EtOH.
- Mix and place at -80°C for 30 min or overnight at -20°C.
- Centrifuge 30min at 4°C. Wash with 70% EtOH and centrifuge for 10min
- Remove supernatant and dry pellets in vacuum desiccator.
- Resuspend in 20µl of TE (pH 8.0) by incubation at 55°C and vortexing.
- Add 5µl of loading buffer before loading onto 0.5×TBE, 0.8% agarose gel (make gel as
long as possible). Electrophorese at 20-40 V during the day (approx. 5hr), or 12 V
overnight.
- Run gel until loading dye is about 2 cm from the bottom. Place UV-fluorescing ruler
beside gel and photograph.
- Place gel in 0.2M HCl for 15min with rocking action to depurinate DNA.
- Decant the HCl and rinse the gel in ddH2O.
- Added denaturation solution and rock for 10min twice.
- Decant the denaturation solution and rinse in ddH2O.
- Added neutralisation solution and rock for 30min.
- Decant the neutralisation solution and rinse in ddH2O.
- Added 2xSSC and rock for 5min before transfer.
- Continue to capillary blotting as on p…. or Posiblot transfer on p….

A note from Andrew: Don’t use root DNA or a pooled sample containing root DNA to digest
for Southerns. It never cuts very well, probably due to inhibitory substances carried thorough
the extraction procedure. All other tissues seem fine…
 16

4. RNA Procedures

RNA tips and tricks

General precautions:
Always wear gloves, not only to protect your samples from RNases, but to
protect you from some of the chemicals.
Pipette tips and eppendorfs should be filled from the RNA stock of tips in the
RNA cupboard. Refill boxes wearing gloves.

DEPC treatment :
All RNA solutions (except Tris containing solutions) and plasticware should
be treated with 0.1%(v/v) of DEPC.
N.B. Do not add DEPC to solutions containing Tris. To make RNase free Tris
solutions, weigh out RNA grade Tris and dissolve in DEPC treated H2O.

Treatment of solutions:
- Put on Gloves
- Add 0.1%(v/v) DEPC to the solution
- Shake very well to disperse the DEPC and incubate overnight at room
temperature
- Autoclave the solution
- After autoclaving and before the solution cools, shake it very well and open the
lid to release the CO2 (otherwise the solution is acidified)

Treatment of plasticware (ie. SS34 tubes):

- Put on Gloves
- Fill the container with water
- Add 0.1%(v/v) DEPC (40µL for a full SS34 tube)
- Shake very well to disperse the DEPC and incubate overnight at room
temperature
- Autoclave the plasticware
- Discard the water

Baking of glassware:
All RNA glassware should be baked at 200°C, overnight. Allow to cool before
use.
 17

Hot phenol RNA extraction Dave

Prepare big tubes with 5mL HB + 2.5mL phenol + 50µL 2mercaptoethanol and put to
65°C just before grinding
Grind 0.5g tissue under liquid N2
Scrape powder into homogenisation buffer/Phenol and mix immediately. Shake 30min RT.
Add 2.5mL Sevag and shake 15min
Pass to 15mL tubes and spin 4.5K RT 15min
Remove aqueous layer and phenol/sevag extract to completion (10min shake/10min spin)
Pass aqueous layer to SS34 tubes. Add 0.1vol 3M NaAc + 2.5 vol ethanol, incubate -70°C
30min
Spin 18K 4°C 30min
Don’t dry. Decant as much as possible and resuspend in 7.5mL H2O, then add 2.5mL 4M
LiCl. Incubate ON at 4°C
Spin 18K 4°C 30min
Transfer the supernatant into a new tube (this contains genomic DNA; see Genomic DNA
processing on p13)
1X 80% ethanol wash
Vac dry and resuspend in 300µL H2O
Pass to eppendorf, add 0.1vol 3M NaAc + 2.5 vol ethanol, incubate -70°C 15min
Spin 13K 4°C 30min
1X 80% ethanol wash
Vac dry and redissolve in 50µL DEPC-H2O
 18

Arabidopsis RNA extraction procedure Tania

♦ Use aluminium foil on bench coat as a work surface
♦ Set waterbath to 60°C
♦ Set up mortar and pestles with liquid N2
♦ Add:
2.5mL phenol
5mL homogenisation buffer
50µl β-mercaptoethanol to a 50 mL RNase free tube and
heat in 60°C waterbath
♦ Weigh out approximately 0.6 g of frozen tissue and add to mortar
♦ Grind tissue to a fine powder whilst continually adding liquid N
♦ Let liquid N2 completely evaporate then add to hot phenol mixture
♦ Shake tube well and place horizontally in shaker for 15 minutes
♦ Add 2.5 mL SEVAG and shake for a further 15 minutes
♦ Transfer mixture to a RNase free 15 mL tube and spin in Jouan at 4500 rpm, room
temp, 15 minutes
♦ Transfer aqueous (upper) layer to a new tube and add 2.5 mL phenol and 2.5 mL
SEVAG, shake 10 min and spin 15 min. This step may need to be repeated a few times
to remove excess protein at interface
♦ Preweigh 50 mL tubes
♦ From this step onwards you need to be particularly careful to avoid RNase
contamination
♦ Transfer aqueous layer to preweighed tubes and weigh (1g ≈ 1mL)
♦ Add 0.1 vol 3M sodium acetate and 2.5 vol ethanol
♦ Incubate at -80°C for 30 minutes (meanwhile reduce temperature of centrifuge)
♦ Spin 4500 rpm, 4°C, 30 min
♦ Remove supernatant and invert tube on a tissue to dry
♦ Resuspend in 7.5 mL DEPC H2O (avoid vortexing if DNA is to be kept)
♦ Add 2.5 mL 8M LiCl (final conc = 2M)
♦ Incubate at 4°C overnight
♦ Transfer to RNase free SS34 tube
♦ Spin 18000 rpm, 4°C, 30 min - 1 hr
- check rotor code, brake switch down all others up
♦ Supernatant contains genomic DNA and this can be kept if needed (see p13)
♦ Wash pellet in 80% ethanol, spin 5 min and remove wash carefully as pellet is very
delicate
♦ Dry in vacuum desiccator
♦ Resuspend in 300µl DEPC H2O and transfer to RNase free eppendorf (if insolubles are
present, spin and transfer to new tube)
♦ Ethanol precipitate RNA: add 0.1 vol 3M sodium acetate and 2.5 vol ethanol, incubate
at -80°C for 15 minutes, spin at 4°C for 30 minutes and wash in 80% ethanol
♦ Dry in vacuum desiccator
♦ Resuspend in 50 µl DEPC H2O
♦ Quantify RNA in spectrometer
 19

The “Pineapple” RNA extraction procedure Tony

This protocol is used for high phenolic, acidic and carbohydrate containing tissue such as
pineapple, mango and papaya fruit tissue.

• Grind tissue (3-6g) under liquid nitrogen in a pre cooled mortar

• Transfer into a SS34 tube containing 2-3 vols of extraction buffer (add 1% 2-
mercaptoethanol before use)
N.B: higher buffer volumes helps remove phenolics and helps buffer highly acidic fruits
such as pineapple
• Vortex for 2 min
• Add 0.25 vols (of tissue and buffer) of ethanol, vortex 1min
• Add 0.11vols (of tissue and buffer) of 5M K Acetate, vortex 1min
• Add 1 vol (of total solution) of SEVAG , vortex 1-2min
• Centrifuge tube at 18,000rpm for 30min at 4°C
• Add 1 vol of SEVAG to aqueous layer , vortex 1-2min centrifuge at 18,000rpm for
30min at 4°C
• Transfer aqueous layer into a falcon tube
• Add 1 vol Phenol-SEVAG and vortex well
• Centrifuge extractions in the Jouan at 4,500rpm for 10min at 4°C
• Repeat this until no interface is seen, usually 3-4 times
• Transfer the aqueous layer into a SS34 tube, and add 2.25vols of ethanol
• Incubate at -20°C for 2hr, then centrifuge at 18,000rpm for 30min at 4°C
• Wash pellet with 80% ethanol, centrifuge at 18,000rpm for 5min at 4°C, air dried
• Resuspend pellet in 10mL DEPC-H2O
• Add 6mL 8M LiCl, incubate O/N at -20°C
• Centrifuge at 18,000rpm for 30min at 4°C
• The supernatant contains genomic DNA, and can be extracted (see p13)
• Wash pellet in 80% ethanol, at 18,000rpm for 15min at 4°C
• Remove ethanol and briefly spin the tubes and remove liquid from the bottom with a
pipette
• Resuspend pellet in 300µL DEPC-H2O, add 0.1vol 3M NaAcetate (pH5.2) and 2vol
ethanol, incubate at -80°C for 30min
• Centrifuge in a microfuge for 30min at 4°C, wash the pellet in 80% ethanol and dry
under a vacuum.
• Resuspend in 50µL DEPC-H2O
 20

RNA electrophoresis Dave

1. Quantify your RNA by A260 (1 A260 unit of single straned RNA = 40 µg/ml). Also
check the A220-320 curve shape and A260/280 ratio. Pure RNA has a 260/280 ration of
2.0, but ratios of 1.7-2.0 are fine. Lower ratios usually mean protein contamination.
2. Run 5-10ug (depending on comb size) on a 1% 0.5X TBE gel for 1 hr at 80V. No
RNase precautions are necessary with the gel, except to keep everything basically
clean , and to scrub the comb and gel tray well with decon.
3. Mix RNA loading buffer and RNA, heat to 65°C for 5min, then chill on ice 3 min before
loading. An volume of loading buffer equal or greater than the sample volume works
well, below this I’m not sure.
4. Run the gel at 80V. Unless separation of bands is required, run the loading dye only
about 5cm from the wells to keep the bands nice and tight.
5. Plants have cytoplasmic 28S and 18S ribosomal RNA bands which allow you to check
that the quantification is accurate There will also be several lower bands which are
chloroplast ribosomal RNAs.
6. Photograph your gel with a ruler.
7. Rock the gel in water for about 10 min in water to get rid of any formaldehyde (stops
transfer) and 10 min in 20XSSC, then set up a capillary transfer as on p23 or Posiblot
transfer as on p25

10x MOPS

20.93g MOPS
2.05g Na Acetate
10mL EDTA (0.5M)
pH 7.2 with NaOH and make volume to 500mL
Add 0.1% DEPC, incubate O.N. and then autoclave

RNA 10X Loading Buffer

750µL Formamide
150µL 10x MOPS
240µL Formaldehyde
130µL water
200µL 50% Glycerol
Add a touch of bromophenol blue powder with a p200 tip to match the colour of an old
aliquot. Store at -80°C
 21

RNA solutions
All solutions except those containing Tris, should be DEPC treated (see RNA tips and
tricks)

Homogenisation buffer (For the Hot Phenol Method)

100mL 400mL
100mM Tris pH 8-9 10mL 40mL of 1M
5mM EDTA pH 8 1mL 4mL of 0.5M
100 mM NaCl 2mL 8mL of 5M
0.5% SDS 5mL 20mL of 10%

Homogenisation buffer (For the Arabidopsis extraction method):

For 100 mL
100 mM Tris (pH8.0) 10 ml of 1M
5 mM EDTA 1 ml of 0.5M
100 mM NaCl 2 ml of 5M
0.5% SDS 5 ml of 10%
82 ml DEPC H2O

Extraction buffer (For the “Pineapple” extraction method)

For 100mL
150mM Tris pH7.5
pH with solid boric acid 15mL of 1M
2% SDS 20mL of 10%
50mM EDTA 10mL of 0.5M

3M Sodium Acetate 5M K Acetate

49.2g in 200 ml

8M LiCl (dangerous!) MOPS buffer (1x)

33.9g in 100 ml 200 mM MOPS
10 mM EDTA
50 mM NaOAc
pH to 7.0 with NaOH

RNA Loading Buffer

750µl formamide
150µl 10x MOPS
240µl formaldehyde
96.8µl H2O
200µl 50% glycerol
4µl 7.7mg/mL EtBr
touch tip to dye and mix, aliquot and store at -80°C

RNA gel (keep everything RNase free)

Use a 1% Agarose in 1x TBE buffer
 22

5. Hybridisation

Radiation Safety
Firstly DON'T PANIC. Radiation work is a useful technique, and if you are
careful, you will be in no danger. Before you do radiation work you must be authorised by
Ng Hock, the botany department radiation officer. After watching the thrilling video on
radiation safety, you will be issued with a radiation safety badge and be allowed to work in
the radiation room. Always get someone responsible (and authorised) to help you the first
time you work in the radiation room.

Do's and Don'ts

Do's
☺ Wear gloves at all time inside the radiation room
☺ Be cautious of radioactive material
☺ Check your work area before and after use
☺ Check equipment before and after use
☺ Clean any contaminated equipment/areas immediately after use
Get someone to show you clean up procedures
☺ Minimise contact with radioactive material, remember the inverse square law,
twice the distance away, you only get 1/4 of the radiation, and so on
☺ Protect vital organs, such as eyes and reproductive organs
☺ Get signed out after each days work
Don'ts
DON'T PANIC
Work without a shield
Contaminate the Geiger counter
Be sloppy
Drink Hyb solution
DON'T PANIC

If you have any questions or concerns, ask a responsible person to help.

 23

Capillary blotting of northerns and Southerns

Preparation of the gel
Southern (DNA) gels:
• Place gel a plastic container and add 0.2M HCl for 20min to depurinate DNA. Place
container on the rocker.
• Decant the HCl and rinse gel in water twice
• Add denaturation solution for 15min with rocking.
• Decant denaturation solution and rinse gel in water twice
• Add neutralisation solution for 30min with rocking
• Decant neutralisation solution and rinse gel in water twice
• Place gel in 2x SSC before transfer

Northern (RNA) gels:

• Place gel in a plastic container
• Add MilliQ water and rock for 10min, this removes any formamide that can inhibit
transfer
• Place gel in 2x SSC before transfer

Capillary blot:
• Cut piece of hybond to the exact size of the gel
• Cut 10 pieces of 3M blotting paper to the same size
• Cut the right hand corner of the gel for orientation
• Mark the same corner on the membrane with a pencil
• Set up the apparatus as follows:

• Immerse wick, membrane and blotting paper in 2X SSC before assembly

• Smooth out each layer, especially those nearest the gel so that no air bubbles exist
• Cover entire assembly with plastic wrap to reduce evaporation
• Place 250g weight on top - use a full 250mL plastic bottle
 24

• Ensure there are no "short circuits" that will draw the liquid away from its correct
path
• Leave overnight where it won't be disturbed
• Disassemble apparatus
• Wash membrane very gently in 2x SSC (leave sit for 30-60 secs with occasional
gentle shaking).
• Dry membrane on a tissue for a few hours
• Fix nucleic acid onto the membrane with the UV crosslinker - do both sides of the
membrane (program C2, 50mJ)
• Store membrane between 2 sheets of clean A4 paper in a plastic bag at room
temperature.

0.2M HCl IL

17.2mL 36% HCl in 1L

20mL 32% HCl in 1L (32% ~ 10M)

Denaturation solution IL

1.5M NaCl 87.66g

0.5M NaOH 20g

Neutralization solution 1L

1.5M NaCl 87.66g

1M Tris HCl 121.14g

pH to 7.4 with lots of HCl

 25

Posi-blot transfer of northerns and Southerns Josh

Not to blot but how to blot, that is the question,
Whether to nobler in the mind to use ancient capillary
or to take arms with the new technology of POSI-BLOT.
All ye kindred, use POSI-BLOT, POSI-BLOT Ra-Ra-Ra !

The POSI-BLOT system uses positive air pressure to force the transfer solution (20X SSC)
from the sponges, through the gels and to transfer the nucleic acids within onto the
Hybond membrane. It has varying degrees of success depending on the nucleic acid size,
thickness and percentage of the gel, depth of gel wells and volume of sample loaded.

PROCEDURE
• Run your northern, Southern gel - try to make the gel as thin as possible and if DNA use
a 0.8% agarose gel and RNA use a 1% gel.
• Treat the gels as in the capillary blotting procedure on p….
• Cut two pieces of 3M paper and one piece of Hybond the same size as the gel
• While the gel is being washed/treated, set up the apparatus
• Place the membrane support grid into the buffer collection base
• Make sure that there is a pre-cut mask (plastic) which is smaller than the gel such that
there is overlap of at least 1mm on all sides (there are precut masks)
• To ensure a good seal make sure the mask is not wrinkled
• Have ready : 20X SSC, Transfer 20X SSC (recycled 20X SSC), sponges, air pump
and connector hose, pencil, membrane forceps, shallow dish (Amersham lid is good),
2X SSC, 3M paper and Hybond.
• Once the gel has been washed/treated in the lunchbox take it to the POSI-BLOT
machine
• Fill the Amersham lid with 20X SSC - Don’t be afraid to squirt this between layers, have
everything wet so that there is less chance of getting air bubbles
• Prewet a piece of 3M paper in 20X SSC and place it on the gel support grid
• Prewet the Hybond and place it on the 3M paper
• Place the plastic mask over the paper and membrane and align such that the
membrane is overlapped evenly on all sides
• Place the gel carefully face up onto the mask - it is important not to move the gel around
too much, try and get it right first go; moving it around to much may cause the mask to
cut the gel edges and it won’t seal.
• Place the other piece of 3M paper on top of the gel and ensure that there are no air
bubbles
• Fill the lunchbox with transfer 20X SSC - Use this to wet the sponges
• Place the sponges on top, red one first so the transfer is fastest and then the other two -
To get an idea of how wet the sponges should be pick it up horizontally from the lunch
box and wait a few seconds. Let any 20X SSC drip off and then apply the sponge.
• Put the lid on and attach the connector hose to the air pump
• With your finger on the end of the hose, switch on the pump and using the knob to
adjust the pressure to 75 mmHg
• Attach the hose to the POSI-BLOT inlet port and stand well back
• Make sure that the pressure goes to 75 mmHg, use adjustment knob if necessary - If
the pressure does not go up there is a leak; the gel may be smaller than the mask, there
 26

may be a cut in the gel edge, the mask could be wrinkled or may have a hole in it, the
fastening clips may not be tight enough or the hose may not be on properly.
• Leave the machine going for at least two hours. Three hours is good and five is
excessive (the pump gets hot)
• Take off the sponges and recycle the SSC
• Remove the 3M on top of the gel
• Mark the wells with a pencil - Push the pencil through the wells and draw the wells on
the Hybond; also mark the bottom right corner (if you have not already done it)
• Check the transfer efficiency on the trans-illuminator later - Southern gels should be
stained as normal while northerns can be checked without staining
• Fill the Amersham lid with 2X SSC and, using forceps, pick up the membrane and rinse
in 2X SSC gently to remove excess salt and gel debris
Place the membrane between two pieces of 3M paper to dry Cross-link the dry membrane
in the UV cross linker using the “C2” program
 27

Northern and Southern Hybridisation

There are 2 different hybridisation buffers, they use different conditions as shown in the
table below.

Church Buffer Formamide Buffer

Prehyb: 65°C 1hour 42°C 4-6hours
Hyb: 65°C overnight 42°C overnight
Buffer composition: 0.5M NaPO4 5mL Formamide
5% (w/v) SDS 2.5mL 20x SSC
(see appendix B) 500µL 10% SDS
200µL boiled salmon tested DNA
(5mg/mL)
1mL Denhardts Solution
800µL ddH2O
N.B Church buffer gives much better results and is easier to make and you would
be unwise to use the other method
-3 -4
Amount RNA 1.67µg 0.167µg 0.0167µg 1x10 µg 1x10 µg

Church Buffer

Formamide Buffer

Prehybridisation
• Turn hyb oven and prewarm hyb-tubes
• Warm up hyb solution
• Wet membrane in 2 x SSC
• Roll membrane up and place in tube with top surface of membrane facing inwards
• Gently pour pre-hyb solution into tube and replace in oven
• Pre-hyb

Preparing Sephadex Columns

• Take a 1mL syringe and pack the bottom tightly with glass wool to a thickness of
approx. 2mm
• Fill syringe to top with sephadex G-50
• Place in 15mL falcon tube and spin down in Jouan at 3000rpm for 1-2 min
• Top up with more sephadex and repeat spin
• Repeat until compacted sephadex fills to the 0.8mL mark
• Wrap ends of syringe with parafilm and store at 4°C until needed
 28

Probe labelling (using the rediprime II kit)

2 half reactions (You cannot make just 1 half reaction with this kit)
• Take 25-50ng of DNA and make the volume to 6µL with TE
• Boil for 10 min and quench on ice
• Resuspend rediprime reaction tube in 33µL TE
• Split into 2 tubes containing 17.5µL each
• Add the DNA into each tube
• Add 2.5µl 32P-dCTP to each tube
• Incubate at 37°C for 30 min
• Cut a 15mL falcon tube off at the 14 mL mark and place an ependorf with the lid
removed in the bottom, place the sephadex column inside the tube
• Add STE to make the probe reaction up to100µl (76.5µl)
• Continue to spin column clean up below

Full reaction
• Take 25-50ng of DNA and make the volume to 45µL with TE
• Boil for 10 min and quench on ice
• Resuspend rediprime reaction tube with the boiled DNA
• Add 5µl 32P-dCTP into tube
• Incubate at 37°C for 30 min
• Cut a 15mL falcon tube off at the 14 mL mark and place an eppendorf with the lid
removed in the bottom, place the sephadex column inside the tube
• Add STE to make the probe reaction up to200µl (150µl)
• Continue to spin column cleanup below
Spin column clean up
• Take a 1µl sample for the scintillation counter then carefully add the rest to the top of
sephadex column
• Spin at 90% for 5 min
• Place eluate into a lock-cap tube
• Take a 1µl sample of the eluate for the scintillation counter
• Do a scintillation count on the two 1µl samples using protocol 11 (ideal is 1million
counts per 1mL of hyb solution)

Hybridisation
• Put small water bath at 100°C
• Place protective cap probe tube and boil for 10 min, quench on ice then spin down
• Add probe to pre-hyb mix avoiding direct contact with membrane
• Replace and hyb overnight (no longer than 20 hours)
• OR, if you are smart, you can denature your probe by just adding 0.75 vol of 2M NaOH
(stock kept in rad room) to probe, mix well, incubate 5min at RT, and add the whole
thing to the hyb bottle, and that’s it.
 29

Washes
• Work out stringency of washes - SDS / SSC concentrations, times and temperatures
Usually the wash conditions are:
2x SSC, 0.1% SDS; 15min/ Room Temperature
2x SSC, 0.1% SDS; 15min/ 50°C
0.2x SSC, 0.1% SDS; 15min/ 50°C

• Make up required solutions and preheat to correct temperatures

• Remove hyb tube from oven and remove the hyb solution into a 32P discard bottle (be
very careful! watch for drips)
• The washes can be done in the hyb bottle or in a plastic tray in the water baths
• Add the wash solutions and agitate continuously for required time and temperature
• First wash should be discarded into waste bottle but second and third depending on
count can be washed down the sink with plenty of running water
• Blot excess water from membrane on 3MM paper then seal in plastic
• Before placing under phosphorimager plate make sure there is no moisture on the
outside
• Place in phosphorimager cassette for 4-6 hours, may be left overnight for a weak signal.

Stripping membranes

• Place membrane into a plastic container

• Boils 500mL of 0.1%SDS
• Add the 0.1% SDS to the membrane
• Allow to cool for 1-2 hours
 30

6. PCR Techniques
Reverse Transcription Polymerase Chain Reaction
Reverse Transcription:
- Mix together:
RT Buffer (10x) 2µL
MgCl2 (25mM) 4µL
dNTP (10mM) 8µL
OligodT/Random Hexamers 1µL
RNA XµL (1µg)
DEPC-H2O up to 18µL

- Incubate
65°C/5min
ice/5min
Room Temperature/10min
- Then add the following:
RNase Inhibitor 1µL
MMLVRT 1µL
Mineral Oil 50µL
- Incubate at room temperature for 10min

- Incubate in a thermocycler with one of the following cycles

Cycle 1 OR Cycle 2
37°C 1hr 42°C 15min
95°C 5min 99°C 5min
4°C hold 5°C 5min

PCR: Using TAQ polymerase

For 1 PCR reaction 1/4 reaction:
- Add the following together:
PCR Buffer (10x) 2.5µL
MgCl2 (25mM) 1.5µL (MgCl2 concentration can be optimised)
dNTP (10mM) 2µL
Primers (15µM) 1.67µL (1µM final concentration)
DNA XµL
H2 O up to 20µL
- Also make a Enzyme mix:
Taq Pol XµL (0.625units)
H2 O up to 5µL
For more than 1 PCR reaction 1/4 reaction:
- If more than one PCR reaction is being prepared make a master mix consisting of buffer,
MgCl2, dNTPs and H2O. Also make a master enzyme mix.
- For a master mix work out how much enzyme, buffer, etc each sample will need and
multiply by .2 more than the number of PCR reactions (eg if you have 5 reactions, multiply
by 5.2).
- Make the master mix and aliquot out into the corresponding tubes, then add the DNA
and primers. The enzyme mix is added later (see below).
 31

Standard Hot Start Program:

Step Step Name Temperature/Time

Number
1 Denaturation 94°C/1min
2 Denaturation 94°C/30sec Cycle steps 2 - 4
3 Annealing 2°C below the lowest melting 35 - 45 times
point of the 2 primers/ 30sec
4 Extension 72°C/ 1min per 1kb based on
predicted product size
5 Final Extension 72°C/15min
6 Chill 4°C/Hold
7 End

- Start the program and pause on step 1. Place all tubes in the PCR machine and allow to
heat to running temperature (94°C).
- Remove each tube and add 5µL of the enzyme mix and 50µL of mineral oil (No need to
add oil for PCR machines with hot bonnet), Place back into the machine. After all tubes
have had enzyme and oil added, press pause again and allow the program to run
normally.

Preparation of 10mM dNTP

Mix together
5µL dATP (100mM)
5µL dTTP (100mM)
5µL dCTP (100mM)
5µL dGTP (100mM)
180µL H2O
 32

Blunt End Cloning of PCR Products

(And how to make CIAP EcoRV pBS) Josh

PART A : CREATING BLUNT ENDED PCR FRAGMENTS

• Run about 20 µl of the PCR reaction on a gel and cut out the band(s) of interest
• GENECLEAN the band using 5µl of glass milk and resuspend in 50 µl H2O
• Fill in the ends as follows :

GENECLEANED band 48 µl
H2 O 38.75 µl
10x Fill in buffer * 10 µl ( * See recipe below)
100mM ATP 1 µl
T4 PNK (10U/µl) 1 µl
Klenow (8U/µl) 1.25 µl
_____
100 µl

• Incubate at 37°C for 1 Hr.

• GENECLEAN the 101 µl using 5 µl of glass milk and resuspending in 13 µl H2O
• Blunt-ended ligation as follows :

GENECLEANED blunt fragment 11µl

0.1 µg/µl CIAP pBS cut with EcoRV 1 µl
10x T4 DNA ligase buffer 2 µl
H2 O 4 µl
T4 DNA ligase (1 u/µl) 1 µl
_____
20 µl

• Incubate at 15°C o/n

• Transform electrocompetant cells with 2µl of ligation reaction and plate out 250µl onto
each of two LB-Amp plates (with X-gal) and incubate o/n at 37oC

* 10X Fill in buffer is :

0.5 M Tris pH 7.5 1.0 mL @ 1 M

0.1 M MgCl2 200 µL @ 1 M
10 mM DTT 20 µL @ 1 M
0.5 µg/µl BSA 100 µL @ 10 µg/µl
200 µM dNTPs 160 µL @ 2500 µM
H2O 520 µL
_____
2.0 mL
 33

PART B : CREATING EcoRV/CIAP TREATED pBS

1. Digestion with EcoRV

pBS 15 µg (e.g. 11.1 µl of 1.37 µg/µl pBS)

10X OPA 24 µl
EcoRV (5 u/µl) 12 µl
H2 O X µl
____
120 µl

• Incubate at 37°C for 2 Hrs

• GENECLEAN using 15 µl of glass milk and elute in 80µl of H2O

2. CIAP treatment of pBS

Digested pBS 79 µl
10X NEB Buffer 3 10 µl
H2 O 7 µl
4 units of CIAP 4 µl (make a 1/10 dilution of 10u/µl CIAP in H2O)
_____
100µl

• Incubate at 37°C for 1 Hr and then terminate the reaction with 1µl of 0.5M EDTA pH 8.0
• Incubate at 75°C for 10 minutes (Optional)
• GENECLEAN using 15 µl of glass milk and elute in 30 µl H2O
• Quantify on a gel using known concentrations of pBS (should get 150 -200 ng/µl)
Dilute to a final concentration of 100 ng/µl using H2O
 34

7. Protein Techniques

Η Fun with PAGE and westerns ΗDave

You can do a PAGE gel too
Getting psyched
• Clean all gel equipment with ethanol and Kimwipes
• Make sure glass plates are completely clean
• Make premix solutions of both stacking and separating gels at the same time
• Make 10% ammonium persulfate fresh weekly (0.05g + 500µL water)
• Stacking gels are always 5%, use 12% separating for beta subunits
• Refer to Tables 18.3 and 18.4 in Blue Book for gel mixes
• A 0.75mm Hoefer mini gel takes about 5mL of separating gel and 2.5mL of stacking
gel. Obviously, double this for 1.5mm combs

Pouring it
• Set up gel casting apparatus: assemble plates with comb fully inserted and mark 1cm
below comb for max level of separating gel
• Add APS and TEMED simultaneously to separating gel mix in a recycled 50mL tube
and vortex. Get a move on and pour it down one side of the plates with a p1000 - you
have 1min before the acrylamide starts to set.
• Overlay carefully with 1mL of ethanol (enough to cover the top of the separating gel by
5mm) because air will prevent acrylamide polymerisation. Allow 30 minutes for
polymerisation.
• Pour off ethanol and rinse 3X with water (turn upside down) and dry around top of gel
with a tissue.
• Position comb on angle and pour stacking gel from the highest side of the comb, then
fix it in place. Allow to polymerise for 20min
• Disassemble the gel setting apparatus and clean off all acrylamide bound to the
outside of the casting plates. Remove comb and rinse wells with gently with water
bottle and drain. Insert gel plates into running apparatus and fill tanks with running
buffer. It is a good idea to clear gunk from the wells by pipetting up and down in them
with a p200. Use the well templates to make loading the gel easier.
• Alternatively, gels can be stored wrapped well in cling wrap and kept in a container
humidifed with some 1X running buffer at 4oC for about 2 weeks (leave the combs in).

Loading
• Prepare you samples: Mix extract with 5X loading buffer. Figure out how many µg you
want to load (10µg total protein works well for beta), what volume to load (10µL is
good for 0.75mm 10 well minicombs) and dilute all your samples to the same
concentration in extraction buffer to the same 1X volume. This allows you to make the
loaded volume for all lanes the same, which makes for a smashing gel.
• Load into the wells. Avoid using the outer lanes as these do not always run properly.
Any spare lanes should be loaded with 1X LB made with the protein buffer if possible
• Run the gel at 100V 2-2.5 hrs or until marker has just run off.
 35

• Once the gel has been run, cut off the bottom right hand corner of the gel as well as the
stacking gel
• Proceed to staining or begin the western

Staining
• Place the gel in stain for 1 hour
• Transfer the gel into destain solution and either place in a shaker at low speed for 2
hours or leave in destain overnight
• After staining a gel the stain solution should be poured back into the stain bottle. Stain
can be re-used many times
• After destaining pour the solution through whatman no. 1 paper containing activated
charcoal. (This will remove all the coomassie blue)
• The charcoal filter can be re-used at least 15 times

Dot blotting proteins

• You can use either nitrocellulose or PVDF. It is much harder to get nice, consistent
dots with PVDF, so I would use nitrocellulose. My version for nitrocellulose is taken
from the Promega Protoblot Western Blot tech manual which also has a method for
PVDF
• Wet the nitrocellulose in 1X TBS and dry thoroughly (20-30min)
• Apply your sample in 1µL volumes to the membrane and allow to dry thoroughly before
proceeding to block in TBST 5% milk (see western method)
• Tip for smart little vegemites: if you want to apply lots of samples, use a template grid
drawn on an OHT sheet on top of a light box with the membrane over that. Face the
ink side of the template down to avoid bleeding and tape down the corners with magic
tape to ensure everything stays still and that the samples go on evenly.
• If you’re doing serial dilutions of extracts, dilute all your samples to a standard
concentration, then use a microtitre plate to make further serials. For 1:2 series, use
50µL + 50µL.

The Western
Are you ready?
• Wash PAGE gel in MQ water to remove excess SDS
• Place gel in transfer buffer for about 20 minutes for 0.75mm gels (removes remaining
SDS) – do ~40 mins for 1.5mm gels.
• Meanwhile, cut the membrane (cut off the bottom right hand corner) plus 2 pieces of
3MM paper (3MM paper provides support). A Hoefer mini gel measures ~ 55 X 85mm.
• Activate PVDF membrane by placing it in methanol 10 secs, then water for 5 minutes
(to remove methanol), then soak membrane and 3MM paper in transfer buffer for 10
minutes
 36

Transfer
• Fill the transfer apparatus with transfer buffer up to the minimum fill level, connect
cooling hoses to a tap and place entire apparatus on a magnetic stirer
• Assemble the transfer cage in a tray containing about 3cm of transfer buffer to keep the
gel and membrane wet. It is important to get positioning of the gel on the membrane
right first time because some protein can transfer immediately and result in ghost
bands if the alignment is changed. Here’s how:

gel membrane

grey cage side

faces anode (+)

black cage side

faces cathode (-)

3mm paper

foam pads

• Insert the cassette (grey facing the red terminal – the anode +). If you’re only
transfering 1-2 gels, stick the cases in the center slots.
• Run for 100 minutes at 100 V for 0.75mm gels, 2 hrs for 1.5mm gels. This will give filth
transfer.
• When completed cut membrane(s) up - cut out marker lanes and stain like a gel if they
are not coloured. Mark blots if multiple antibodies are used
• Wash membrane in MQ water
• Start blocking remaining membranes in TBST 5% milk O/N. on a rocker at 4oC. Can do
at RT for 1 hour, but O/N. is better
• OR dry the membrane on filter paper. Note: If the membrane is dried it must be re-wet
by placing it in methanol for 10 seconds and than washed in MQ water for 5 minutes
 37

All subsequent steps at RT on rocker

• In the morning, rinse once and then wash 3X 5 min TBST 1% milk.
• Replace with 10mL TBST 1% milk containing 1° Ab (1/1000 to 1/100. For tgb1 Ab, use
7.5µL in 10mL – i.e. ~1/1300 dilution) and incubate for up to 3 hrs (30min is usually
OK)
• Repeat above TBST 1% milk wash
• Replace with 10mL TBST 1% milk containing 2° antibody (1/5000 dilution of 1mg/mL
Promega anti-rabbit AP conjugate stock) and incubate up to 2 hrs (30min is usually
OK)
• Wash 3X 5min in straight TBST
• Wash in straight colour buffer 5min, dump, then add 10mL colour buffer with substrates
(33µL and 15µL of Promega 50mg/mL stock respectively). Colour should develop
within a few minutes
• Terminate the reaction by washing in MQ water and then dry on absorbant paper

Notes on Membranes

• Membranes for protein work are more suceptable to damage and therefore extra care
is needed to avoid scratching the membrane
• Hybond-C pure -Pure unsupported nitrocellulose, fragile
• PVDF - Hydrophobic (requires methanol to wet the membrane), High mechanical
strength (less fragile), transfer proteins to the smooth side of the membrane

PAGE and western blotting solutions

Acrylamide 30% stock 100mL

Acrylamide 29.1g
Bis-acrylamide 0.9g

Of course, acrylamide is filthy toxic and you should be a little paranoid. Stock is good
wrapped in foil at 4ºC for anywhere between 1 and 3 months

5X Tris-Glycine running buffer pH 8.3 1L

125mM Tris pH 8.3 15.1g
1.25M Glycine (electrophoresis grade) 93.8g
0.5% SDS 50mL @10%

Note that the grade of SDS greatly affects running of the gels. Find a good grade and stick
to it.

1M Tris pH 6.8 1.5M Tris pH 8.8

30.3g Tris 45.4g Tris
pH 6.8 with HCl pH 8.8 with HCl
Make Volume to 250mL Make Volume to 250mL
 38

2X SDS loading buffer 10mL

100mM Tris pH 6.8 1mL @1M

4% SDS 4mL @10%
0.1% bromophenol blue touch the tip
20% glycerol 2mL
0.2 M DTT 2mL @1M

Dispense into 300µl aliquots and freeze at -20oC

Stain Destain
1.25g Coomassie blue 225mL Methanol
225mL Methanol 225mL H2O
225mL H2O 50mL glacial acetic acid
50mL glacial acetic acid

Transfer buffer 2L

25mM Tris 6.1g

192mM Glycine (electrophoresis grade) 28.82g
15% high grade methanol 300mL

Do not pH and remember that methanol is a carcinogen.

TBST 1L 2L
10mM Tris 1.21g 2.42
150mM NaCl 8.77g 17.53
0.05% Tween 20 0.5mL 1.0

pH to 7.5
plus 1 or 5% (w/v) milk powder. Diploma skim milk powder works well.

Colour Buffer (for Alkaline phosphatase) 1L

100mM Tris 12.12g

100mM NaCl 5.85g
5mMMgCl2 1.02g (MgCl2.6H2O)

pH to 9.5
 39

ELISA
Notes:
• Once the antigen has been bound to the ELISA plates, it will not come off. The plates
can be handled minimum care.
• It is important not to let the plates dry out. When doing the wash steps, only do one
plate at a time.

ELISA
• Dilute antigen to 1µg/ml in binding buffer
• Using a multi-dispense pipette, aliquot 100µl of diluted antigen into each test well of a
96 well, high binding capacity, ELISA plate. (remember to do duplicates of each sample)
• For the blank, load 100µl of binding buffer into 2 wells
• For the positive control, load 100µl of 1 in 1000 diluted 4mg/ml IgG (dilute in binding
buffer)
• Cover the wells with a piece of Contact (sticker)
• Wrap the plates in glad wrap and store at 4oC overnight.
• Tip the solution out of the wells and submerge the plates in water
• Tip out the water and submerge the plates again in the water.
• Tip out the water
• Pour Blotto into the wells and then tip it out (repeat once more)
• Pour Blotto into the wells, so that every well is completely full
• Leave at room temperature for 1 hour.
• Note: this is a good time to make up the antibody dilutions (it usually takes about
1 hour to do)
• Wash the plate once in water and 3 times in washing buffer
• Note: keep the run off from the washing buffer as it can be used later on
• Add 100µl of each antibody dilution into 2 well
• Note: Add 100µl of antibody dilution buffer to the Negative and Positive control
• Incubate at room temperature for 3 hours
• Wash the plates once with water and then 3 times with washing buffer
• Add 100µl of 0.1µg/ml biotinylated anti-rabbit IgG F(ab’)2 fragment (1 in 5000 dilution,
use antibody dilution buffer) into each well
• Incubate at room temperature for 2 hours
• Wash the plates once with water and then 3 times with washing buffer
• Add 100µl of 0.05U/ml Streptavidin bound horse raddish peroxidase (1 in 10,000
dilution, use antibody dilution buffer) into each well
• Incubate room temperature for 30 minutes
• Wash the plates once with water and then 3 times with washing buffer
• Add 100µl of OPD solution into each well
• Incubate at room temperature for 20 minutes
• Read the absorbance of each well at 492nm using the negative controls as blanks
 40

ELISA solutions
0.1M NaPi (pH 7.4) (2 Litres)
5.928g NaH2PO4.2H2O
23g Na2HPO4

Washing buffer (4 Litres)

• Soak 2g of gelatin in ~20mL of water for 3 minutes
• Add gelatin to ~100ml of boiling water
• Keep stiring on a hot plate until the gelatin is disolved
• Add 4ml of Tween-20
• Add 14.5g NaCl
• Add 1 Litres of 0.1M NaPi

Antibody buffer (500 ml)

Disolve 0.5g of gelatin is disolved as in washing buffer
Add 125ml 0.1M NaPi

Blotto (500ml)
25g Powdered milk
150µl Antifoam A
make up to 500ml with PBS
(make up fresh)

OPD solution (25ml)

Note: OPD is TOXIC, wear mask and gloves

• Weigh out ~10mg (5 -20mg is okay) of O-Pheylene diamine (OPD)

• Add 24mL acetate buffer to OPD chips
• Add 3µl hydrogen peroxide
• Use a spatula to break up OPD chips
• Wrap the 50mL tube in alfoil to prevent light exposure and vortex the solution
 41

Bradford assays Matt

Materials
thawed, mixed and spun samples on ice
microtitre plate, wells with flat bottoms
designation sheet for wells
p1000, p200 and p10 and tips
multiwell pipettor
10mg/mL BSA stock
water
Biorad dye concentrate
pipette box lids for water and for dye
bunsen burner
burner lighter

Method
• Make a 0.2mg/mL standard stock, by adding 20µl of 10mg/mL BSA to 980µl of MQ.
Vortex.
• Use four standards: 0.01 0.02, 0.04 and 0.06mg/mL, plus a dye blank IN DUPLICATE.
• Based on a total well volume before adding dye of 200µl, the amount needed for the
0.01mg/mL standard is 0.2 x X = 0.01 x 200; X being 10µl. Similarly, 20, 40 and 60µl
are needed for the 0.02, 0.04, and 0.06mg/mL standards, respectively.
• To get an idea of the amount of sample to add per well, use an unused corner of the
plate. Make up the 0.01 and 0.06mg/mL standards side by side in a total of 200µl. To
another well add, say, 10µl of sample to 190µl of MQ. Add 40µl of dye to each well
and mix well. The colour intensity of the sample must be between the lowest and
highest standard. Adjust the sample volume accordingly. It is prudent to test for a few
samples to get an idea of the range. Also, leaves normally have much more protein
than roots (in 200µl total well vol. + 40µl dye, might typically need 2-5µl for leaves, 10-
20µl for roots, assuming a 5:1 extraction buffer to tissue FW ratio).
• It is important to include in the dye blank and standards the same volume of extraction
buffer as the volume of extract loaded in the sample wells. For example, if 10µl of each
sample is used in the sample wells, then 10µl of extraction buffer must be added to the
dye blank and standards.
• the multiwell pipettor is useful to dispense the MQ for the sample wells.

Blank and standard volumes for 10µl of sample:

MQ (µl) Extraction buffer 0.02mg/mL BSA
(µl) (µl)
Blank 190 10 -
0.01 180 10 10
0.02 170 10 20
0.04 150 10 40
0.06 130 10 60
 42

For 5ul of sample:

MQ (µl) Extraction buffer (µl) 0.02mg/mL BSA (µl)
Blank 195 5 -
0.01 185 5 10
0.02 175 5 20
0.04 155 5 40
0.06 135 5 60

• After the dye blank, standards and samples are loaded IN DUPLICATE, add 40µl of
dye to each well. Use the multiwell pipettor and mix well. Then flame with a bunsen
burner to remove bubbles. Proceed to plate reader and read plate.
 43

8. Plant tissue culture and transformation

Agrobacterium tri-parental mating (updated by Josh 25 th

Nov ‘98)

Tri-parental mating uses natural conjugation between bacteria to transfer a plasmid from
E.coli (donor) to Agrobacterium (recipient) with the conjugal assistance from (helper) E.coli
(HB101/pRK2013). Successful conjugation is selected for by combinatorial selection with
antibiotics. Advantages it has over electroporation are:

1. No electrocompetent cells need to be made

2. There is no chance of molecular re-arrangements that can occur when electroporating
high molecular weight plasmids into Agrobacterium

Notes on Agrobacterium media:

There are a number of possible media that can be used Agrobacterium growth including
(MinimalA, LB, YEP, YEB, YM etc). Some of these media provide additional selection for
Agrobacterium by using sucrose as the primary carbon source rather than glucose. E.coli
cannot metabolise sucrose well. The following procedures all involve use of LB for
Agrobacterium growth.

Procedure:

ϑ Make plates for donor Agrobacterium (LBA4404: LB-Rif50/Strep25 or GV3101: LB-

Rif50/Gen25 - Rif50 alone is O.K.)
ϑ Make a plate for donor (e.g. LB-Tet10 for pAOV, pSOV - plates depend on the vector
you are using) and for helper (LB-Kan50) as well a LB (only) plate for the mating.
ϑ Make a plate that contains antibiotics for your vector as well as for the Agrobacterium
(e.g. LB-Rif50/Tet2)
ϑ Grow the bacteria on the plates (start Agrobacterium first) until good sized individual
colonies are present
ϑ Use a loop to take a colony from each plate and mix well in the middle of the LB plate
ϑ Leave at 28°C for 24 hours
ϑ Take a loop of the mix and plate out (16 streak) onto the combinatorial selection plate
ϑ Place in 28°C for 2-3 days and then pick an isolated individual colony for culture (use
some of the bacteria left to PCR screen the Agrobacterium - see “PCR Screening of
Agrobacterium” in Plant Genetic Engineering Lab Protocols) – Note it is important to
“ease” the bacteria back onto selection (LB-Rif50/Tet2/Strep25 or Gen25 all at once
after mating is too strong and no colonies will grow back from the mating).
ϑ You can restreak a chosen colony onto stronger selection before picking to colony for
growth and PCR (e.g. LB-Rif100/Tet2/Strep25)
 44

Control plate:
If you want to check the tri-parental mating (usually if you have
never done it before) you can have a control plate – i.e. LB plate DR DH
with only two of D,H or R (D – donor; H – helper; R – recipient)
RH
ϑ To save on plates, mix DR, DH and RH in three separate parts
of a LB plate (as shown on right)
ϑ Leave at 28°C for 24 hours
ϑ Take a loop of the mixes and plate out (16 streak) onto a
combinatorial selection plate
ϑ Place in 28°C for 2-3 days – you should get no colonies (you
may get a few colonies from the DR – low rate of conjugation).
This tests that your helper helps and that the combinatorial
selection is good.
 45

Arabidopsis vacuum infiltration (in planta)

transformation (updated 24APR’98 by Josh)
Arabidopsis preparation
• Fill up an 8cm diameter pot with a fairly moist soil mixture and make sure that the soil is
a little above the level of the top of the pot
• Cover the pot with 15cm diameter (flyscreen) mesh circle, and use a size 28 rubber
band to secure it, keeping the mesh taught across the top, and making sure that the soil
touches the mesh
• Spray 2g/L Mancozeb (Yates Mancozeb Plus Garden Fungicide) onto the soil
• Sprinkle Arabidopsis (ecotype Columbia) – approximately 20 or so per pot
• Place the pots into a Yates seedling chamber (holds ~16), put on the lid and place the
containers in the 4°C cold room for 3-5 days (longer if you want) for vernalisation (the
seeds require a cold snap for effective germination).
• Put the plants under short day lights (ie. 8hr day) for 10-14 days and over the last few
days, open the vents and then remove the covers (short day encourages vegetative
growth)
• Thin the seedlings out if they need it – planting them well in the first place is easier in
the long run
• While the plants are in the chambers, they don’t need much (if any) additional water.
Once out of the chambers, water every 2-3 days.
• Place the pots in long day (ie. 16hr day) to induce bolting. Prune back bolts if
necessary to encourage numerous unopened buds at time of infiltration (clip primary
inflorescence at its base, letting secondary inflorescences grow until they start to show
open flowers and these bolts can also be clipped if Agro strains are not ready). I have
had success transforming more mature plants and apparently you can even transform
non-bolted plants.
Π Make sure the plants are not water stressed or their stomata will be shut

Agrobacterium preparation
• Just as the plants begin to bolt plate out your Agro-pFIN/pSOV onto YEB-Rif50Tet2 at
28°C
• In the morning, start a 3.5ml YEB-Rif50Tet2 culture with fresh Agro from the plates and
shake at 28°C for 2 days (start >1 culture for each construct as not all will start)
• At about midday, add what should be a thick Agro culture to a 100ml Erlenmeyer
containing 35ml YEB-Rif50Tet2 and after shaking that overnight, add the thick culture to
a 1L Erlenmeyer containing 400ml YEB-Tet2 (No Rif). Grow these at 28°C for about
24hrs or a few hours longer if possible. (Should make Agro late log/early stationary
phase)

Infiltration Day
Π Sterility is not necessary for these procedures.
• Pellet the Agro culture (ideal OD600 is about 1.6-2.0) in the GSA rotor at 5K for 20mins
at room temperature (although if the centrifuge is at 4°C the infiltration still works).
• Resuspend the cells in ~1L infiltration media (to save labour, pour about 50ml infiltration
media into each bottle and put into shaking incubator to resuspend the Agro. pellet, then
make the culture to a final OD600 of 0.8 (give or take about 0.1) with infiltration media).
 46

Infiltration
• Switch the pump on and close off the vacuum ball valve so the pump is pulling a
vacuum against the vacuum ball valve only.
• Let the pump warm up for a few minutes (peak performance is at 50-70oC – but this
temperature it is not crucial)
Π If you are using this for the first time: GET HELP! This is a very expensive piece
of equipment and you must find out exactly how to use it before you start.
• You will need:
ϑ 5 pots of plants - in a large plastic container for storage afterwards
ϑ 2 wooden skewers (break off to 2/3 normal length)
ϑ the culture - in the 1L (or larger) plastic beaker
ϑ 1L soft drink bottles (with the top half cut off)
ϑ the vacuum chamber (with hose and bung)
ϑ masking tape (or labels) and pen for labelling each pot
ϑ old Milo or coffee tin for waste (is covered with alfoil and taken to autoclave)
• Pour some of the culture into two of the soft drink bottles and put them into the chamber
• Put a skewer through opposite holes in the bottom of two pots and invert them in the
bottles. Each pot should be suspended upside-down in a bottle with the skewer holding
the pot a few cm’s from the bottom
• Ensure all the plants are covered by the culture (especially the bolt and flowers)
• Put the lid on the chamber, close off the air outlet on the chamber, open the vacuum
ball valve and draw a vacuum until solution bubbles vigorously then shut the vacuum
ball valve and release the vacuum as quickly as possible. DO NOT SWITCH OFF THE
PUMP (constant stop/start of the pump is bad for it – use the vacuum ball valve)
Π If the solution doesn’t boil vigorously then you have a leak somewhere in the
apparatus. Try listening for a hissing sound which will be air being sucked in
through the leak
Π You can use the same Agro for two pots but fresh is best
Π You can tap the sides of the chamber to help “shake” air bubbles off the plant
Π If you watch the leaves within the culture you will see them darken when the
vacuum is released - this is the Agro culture replacing the air spaces in the plant
• Repeat the infiltration procedure to the same plants (no need to remove lid of chamber)
• If you are doing repeated infiltrations, you can save time and wash-up by rinsing all left
over Agro into the waste container and then spraying the beaker, GSA tubes and soft
drink bottles with EtOH. You rinse off the EtOH with hot water and let them drain ready
for the next infiltration (no need to clean chamber between infiltrations)
• Place the plants on their sides in the plastic container, wipe dry the side of the pot and
put a label with the construct name on it then cover the top of the container with Glad-
wrap
• Place the containers under long day light for just one night. The next day remove the
Glad-wrap and set plants upright
• Do NOT water the plants until approximately 6 days after infiltration (although keep an
eye on the plants - water when they are looking wilted)
• Grow plants until bolts are starting to yellow on some siliques and put seed catchers on
them
 47

ϑ Seed catchers are made from soft-drink bottles by cutting off the bottom and
making a hole in the bottle near the neck (about where the curve begins to
straighten out into the cylinder of the bottle)
• When you have enough seed, stop watering the plants and when the entire plant is dry,
harvest the seeds (seeds from green siliques contain germination inhibitors)

Basta® selection of transformants

• Plant ~0.5ml of seed in a seedling tray containing moist soil, put the chamber lid on then
put at 4°C to vernalise (3-5 days)
• Put the chambers under LONG day light and at emergence, spray the plants with
1000mg/L glufosinate ammonium (active component of Basta® – is at a concentration
of 200g/L in Hoechst Horticulture Basta® U.N. No 2902 obtained from Dr Y. Sharma,
AGREVO P/L, 44 Thomas Rd, Kwinana 6167 through Jennifer of AGREVO (03) 9248
6625) – ie. 5ml Basta per litre gives working concentration
• Spray the plants three days later and again three days after that. The transgenics will
be very green and much bigger than the non-transgenics which should wither and die.
You can later transfer the transgenics to individual pots if you so desire
Π You can expect 75-100 transformants from five plants with this procedure

Solutions and Media

Infiltration media 1 litre 2 litres 5 litres

0.5x MS salts 2.16g 4.32g 10.8g
5% w/v Sucrose 50g 100g 250g
1x B5 (or MS) vitamins 1ml@1000X 2ml@1000X 5ml@1000X
0.25g/L MES 0.25g 0.5g 1.25g
PH to 5.7 (with KOH)
After a
Utoclaving add:
10µl/L of 1mg/ml BAP
50µl/L Silwet L-77
(detergent)

YEB 500ml 1L 2L
10g/L peptone 5g 10g 20g
1g/L yeast extract 0.5g 1g 2g
5g/L Sucrose 2.5g 5g 10g
0.5g/L NaCl 0.25g 0.5g 1g
PH to 7.2
 48

Arabidopsis floral dip transformation protocol

(Straight from http://www.cropsci.uiuc.edu/~a-bent/protocol.html by Dave on 21/01/00. Dave and Andrew’s highly
useful extra notes are added in bold italics)

Steve Clough and Andrew Bent, University of Illinois at Urbana-Champaign.

Our present protocol (Clough and Bent, 1998; modified from Bechtold et al. 1993) is
extremely simple. We have found that the MS salts, hormone, etc. make no difference,
that OD of bacteria doesn’t make much of a difference, that vacuum doesn’t even make
much of a difference as long as you have a decent amount of surfactant present. Plant
health is still a major factor - healthy fecund plants make a big difference! With this method
you should be able to achieve transformation rates above 1% (one transformant for every
100 seed harvested from Agrobacterium-treated plants).

Grow healthy Arabidopsis plants until they are flowering. Grow under long days in pots in
soil covered with bridal veil, window screen or cheesecloth.
(Optional) Clip first bolts to encourage proliferation of many secondary bolts. Plants will be
ready roughly 4-6 days after clipping. Clipping can be repeated to delay plants. Optimal
plants have many immature flower clusters and not many fertilized siliques, although a
range of plant stages can be successfully transformed.
Prepare Agrobacterium tumefaciens strain carrying gene of interest on a binary vector.
Grow a large liquid culture @ 28oC in LB with antibiotics to select for the binary plasmid,
or grow in other media. You can use mid-log cells or a recently stationary culture. This
works well for 4 pots: pick a single colony from a selective plate to a 2ml selective
starter (e.g. Tet2 Rif50 Str25 for strong selection of Basta binaries), grow about 24
hrs, innoculate 200ml final culture (Use Tet2 only – Rif is toxic to plants), grow for
~18-24 hrs. This will gives an undiluted A600 (water blank) of something like 1.6,
which means will will get 400mL of Agros after resuspending in the infiltration
medium, and 100ml per pot is perfect.
1. Spin down Agrobacterium (5 min @ 5K using GSA rotor), resuspend to OD600 = 0.8
(can be higher or lower) in 5% Sucrose solution (if made fresh, there’s no need to
autoclave. Ordinary white sugar is fine). You will need 250mL (approx.) for sixteen
3.5” (9cm) pots.
2. Before dipping, add Silwet L-77 to a concentration of 0.05% (500µl per 1L; 125µL per
250mL) and mix well. If there are problems with L-77 toxicity, use 0.02% or as low as
0.005%.
3. Dip above-ground parts of plant in Agrobacterium solution for 2 to 3 seconds, with
gentle agitation. You should then see a film of liquid coating plant. Some investigators
 49

dip inflorescence only, while others also dip rosette to hit the shorter axillary
inflorescences.
4. Place dipped plants under a dome or cover for 16 to 24 hours to maintain high humidity
(plants can be laid on their side if necessary). Do not expose to excessive sunlight (air
under dome can get hot).
5. Water and grow plants normally, tying up loose bolts with wax paper, tape, stakes,
twist-ties, or other means. Stop watering as seeds become mature.
6. Harvest dry seed. Transformants are usually all independent, but are guaranteed to be
independent if they come off of separate plants.
7. Select for transformants using antibiotic or herbicide selectable marker. For example,
vapor-phase sterilize and plate 40 mg = 2000 seed (resuspended in 4 ml 0.1%
agarose) on 0.5X MS/0.8% tissue culture Agar plates with 50 ug/ml Kanamycin, cold
treat for 2 days, and grow under continuous light (50-100 microEinsteins) for 7-10 days.
8. Transplant putative transformants to soil. Grow, test, and use! For higher rates of
transformation, plants may be dipped two or three times at seven day intervals. We
suggest one dip two days after clipping, and a second dip one week later. Do not dip
less than 6 days apart.
References:
Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium-mediated gene
transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris, Life
Sciences 316:1194-1199.
Clough SJ and Bent AF, 1998. Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana. Plant J 16:735-43.
Additional commentary can be found by searching the Arabidopsis newsgroup archives:
http://genome-www.stanford.edu/cgi-bin/biosci_arabidopsis
 50

Mung bean transformation

Mungbean seeds (cv. Burnett) were placed in a darkened container of aerated water
overnight. Seeds were then rinsed extensively to remove germination inhibitors, planted in
trays of soil and placed in a growth cabinet at 25°C with a 16h photoperiod. The first
expanded leaves were harvested and placed on moist filter paper prior to bombardment.

Preparation of DNA coated particles:

Tungsten Particles (0.7µm):
Resuspend approximately 20mg in 200µL ethanol (5mg/shot), vortex for 10min.
Centrifuge and wash in ethanol 3 times, then 3 times in sterile ddH2O.
Resuspend to a final concentration of 100µg/µL in ddH2O.

Prepared DNA:
Added together:
50µL Tungstein (100µg/µL)
20µL DNA (2µL/µg)
50µL CaCl2 (2.5mM) } Add both of
20µL Spermidine (100mM) } these in quick succession

Allow to sit for 5min, to collect tungstein on the bottom, remove approx 10µL from the top
and discard. This gives enough for 5 shots. Do shots as quickly as possible as DNA
dissociates from tungsten.
Prior to shooting swab the gun and the bench with alcohol
Shooting:
Resuspend tungsten-DNA thoroughly and pipetted 4µL into the filter units (use same filter
and baffle for each group of 5 shots)
Working aseptically place baffle onto the media containing the tissue, slightly press into
the agar.
Screw the filter into the gun
Place tissue (on media with baffle) into the gun
Evacuated the gun chamber until the vacuum gauge reads -29mmHg
Press the fire button.
Immediately release the vacuum and remove tissue.
In between shots wipe down the inner chamber with ethanol.

Conditions for shooting:

Helium Pressure: 1000KPa
Distance target to filter: 15cm
Pulse time: 50msec

Leaves were placed in the growth cabinet for 24 hours following bombardment before
being histochemically assayed for GUS activity as described previously.
 51

Papaya Transformation Tony

(Growing temperature 22-25°C)

Somatic Embryo Induction

Cut out embryos from immature (90 days old) papaya seeds.
Culture on somatic embryo induction medium (SEIM) for 1-2 months or 3-4 months
(slow growing embryos).
Sub culture every 2 weeks on fresh SEIM.

Squashing
7-12 embryos are squashed (using a metal spatula) on 3MM filter paper, leave on
SEIM for 1 months. 3 days before shooting, the embryos are subdivided and
squashed.

Bombardment
Place embryos (still on the filter paper) onto osmoticum medium (OSM) in the
morning. At least 4hr before shooting, and keep on OSM for 8hr before, during and
after shooting.
Conditions for shooting:
Pressure: 1000KPa
Distance target to filter: 12.5cm
Pulse time: 50msec

Tungsten Particles (0.7µm):

Resuspend approximately 20mg in 200µL ethanol (5mg/shot), vortex for
10min. Centrifuge and wash in ethanol 3 times, then 3 times in sterile ddH2O.
Resuspend to a final concentration of 100µg/µL in ddH2O.

Prepared DNA:
Added together:
50µL Tungstein (100µg/µL)
20µL DNA (approx 5µg total)
50µL CaCl2 (2.5mM) } Add both of
20µL Spermidine (100mM) } these in quick succession
Allow to sit for 5min, to collect tungstein on the bottom, remove approx 110µl from
the top and discard. This gives enough for 5 shots. Do shots as quickly as possible
as DNA dissociates from tungsten.

Prior to shooting swab the gun and the bench with alcohol
Shooting:
Resuspend tungsten-DNA thoroughly and pipetted 4µL into the filter units (use
same filter and baffle for each group of 5 shots)
Working aseptically place baffle onto the media containing the tissue, slightly press
into the agar.
Screw the filter into the gun
Place tissue (on media with baffle) into the gun
Evacuated the gun chamber until the vacuum gauge reads -29mmHg
Press the fire button.
Immediately release the vacuum and remove tissue.
In between shots wipe down the inner chamber with ethanol.
 52

Recovery
Place embryos (still on the filter paper) onto recovery medium (RM) after shooting
for 5 - 7 days.

Selection
Place all embryos onto FSM media with 150mg/L Kanamycin, for 3 months,
subculture every 2 weeks.
Place tissue onto to FSM with 300mg/L Kanamycin for 1 month Surviving tissue is
placed onto SEIM without Kanamycin for 1 month

Regeneration
A. Embryo germination.
Place onto embryo germination medium (EGM) with 75mg/L kanamycin for
3-4 months (or longer until germinating clumps emerge).
Sub culture every 2 weeks.
Leave on until you see germinating tissue (roots ad shoots).
If the tissue is not germinating, it may have been in tissue culture for too
long, place the tissue on plain ½ MS media to recover the embryos.
B. Single shoot growing.
Place green tissue onto full strength single shoot growing (SSGM) until you
get a whole plant.
Subculture every month.

Micropropropagation
A. Shoot multiplication.
Cut up stems: Cut in between nodes if far apart or in between every second
node if close together. Remove the leaves and roots. Place onto shoot
multiplication medium (SMM) for 1 month.
 53

B. Root Induction.
Cut new emerging shoots (over 1cm+) from central shoot and place onto root
induction medium(RIM) for 3 days.
C. Place shoots onto full strength SSGM. Subculture every month
until a full grown plant ready for potting out.
D. The plant can be kept longer (up to one year) in culture using a
minimal growth medium containing full strength SSGM plus 1%
fructose instead of glucose. Sub culture as needed.

Potting out
A. Plant out 3-4cm plants with root primordia or roots no longer than 1cm. The
plantlets were potted into peat pots, using fresh soil. 2 days prior to
introduction of the plants into the glasshouse, it was sprayed with diazanon.
These plants are to be placed in a humidifying chamber with the following
acclimatisation conditions:
1st week 90-100% humidity
2nd week 70% humidity
3rd week 60% humidity
4th week open door a bit
Leave in chamber until leaves become shiny. Plants should be gently
watered with distilled water when needed. The chamber should be inside the
greenhouse and should be on a sunny spot.

B. Transfer peat pots into new pots and repot as needed

Troubleshooting
Shoots failing to elongate:
Place the stems into elongation media (ELON) for up to 3 months,
subculturing every month.
New emerging shoots should be placed into SSGM media
 54

Papaya media
1. Somatic embryo induction media (SEIM) 1L
1/2 strength MS salts 2.17g
MS Vitamins 1mL (1000x stock)
2,4-D 10mL (1mg/mL stock)
Glutamine 400mg
Myo inositol 10mL (1mg/mL stock)
thiamine HCl 10mL (1mg/mL stock)
Sucrose 60g
Agar 8g
or Phytagel 5g
pH 5.65 - 5.7

2. Osmoticum media (OSM) 1L

1/2 strength MS salts 2.17g
1/2 MS Vitamins 500µL (1000x stock)
Mannitol 36.4g
Agar 8g
or Phytagel 5g
pH 5.65 - 5.7

3. Recovery media (RM) 1L

1/2 strength MS salts 2.17g
1/2 MS Vitamins 500µL (1000x stock)
Sucrose 30g
Agar 8g
or Phytagel 5g
pH 5.65 - 5.7

4. Full selection media (FSM)

SEIM with 150mg/L Kanamycin (1500µL of a 100mg/mL stock in 1L)
or 300mg/mL Kanamycin (3000µL of a 100mg/mL stock in 1L)

5. Embryo germination media (EGM) 1L

1/2 Strength MS salts 2.17g
1/2 MS Vitamins 500µL (1000x stock)
Kinetin 54µL (1mg/mL stock)
IAA 787µL (1mg/mL stock)
GA3 0.8µM (8mL of a 100µM stock, filter sterilised, added
after autoclaving)
Sucrose 30g
Agar 8g
or Phytagel 5g
pH 5.65 - 5.7
 55

6. Single shoot growing media (SSGM) 1L

MS-salts 4.34g
De Fossard’s Vitamins 50mL (2X)
Sucrose 30g
Agar 8g
or Phytagel 5g
pH 5.65 – 5.7

7. Shoot multiplication media (SMM) 1L

MS-salts 4.34g
De Fossard’s Vitamins 50mL (2X)
Sucrose 30g
1µM Kinetin 800µL (1mg/mL stock)
1µM NAA 186µL (1mg/mL stock)
Agar 8g
or Phytagel 5g
pH 5.65 - 5.7

N.B Originally BAP was used as below

1µM BAP - 226µL (1mg/mL stock)

8. Eongation Media (ELON) 1L

MS-salts 4.34g
De Fossard’s Vitamins 50mL (2X)
Sucrose 30g
NAA 200µL (1mg/mL stock)
GA3 1mg/L (29mL of a 100µM stock, filter sterilised, added
after autoclaving)
Agar 8g
or Phytagel 5g
pH 5.65 - 5.7

9. Root induction media (RIM) 1L

MS-salts 2.17g
De Fossard’s Vitamins* 50mL (1X)
Sucrose 30g
10 µM IBA 2mL (1mg/mL stock)
Agar 8g
or Phytagel 5g
pH 5.65 - 5.7

*De Fossards vitamins with no riboflavin

 56

Vitamins #6 (2X) 2L
Inositol 4.32g
Nicotinic acids 196mg
Pyridoxine HCl (100mg/mL) 496µL
Thiamine HCl 539mg
Biotin (50mg/mL) 200µL
Folic acid (50mg/mL) 712µL
Ca-Pantothenate (50mg/mL) 1910µL
Riboflavin 150.8mg (heat to dissolve)
Ascorbic acid (100mg/mL) 704µL
Choline chloride (100mg/mL) 560µL
Glycine (100mg/mL) 1504µL
L-Cysteine HCl 756mg

Vitamin Stocks
Pyridoxine HCl (100mg/mL) 1.5g/15mL in H2O/ETOH
Biotin (50mg/mL) 750mg/15mL dil HCl
Folic acid (50mg/mL) 750mg/15mL dil NaOH
Ca-Pantothenate (50mg/mL) 750mL/15mL H2O
Ascorbic acid (100mg/mL) 1.5g/15mL H2O
Choline chloride (100mg/mL) 1.5g/15mL H2O
Glycine (100mg/mL) 1.5g/15mL H2O

Hormone Stocks
Hormone Dissolve in Concentration of Stock
BAP 1M HCl 1mg/mL
NAA 1M NaOH 1mg/mL
IAA 1M NaOH 1mg/mL
GA3 1M NaOH 34.6mg/L (100µM)
Kinetin 1M HCl 1mg/mL
2,4-D ETHOH 1mg/mL
IBA 1M NaOH 1mg/mL
 57

Tobacco seed sterilisation Dave

Choose either the bleach/ethanol method or the chlorine gas nuke-‘em method. The
chlorine gas way is better for bad contamination or large amounts of seed (it’s quicker).

Bleach/Ethanol Method

• Put the seeds in 1.5ml tubes.

• Add 1mL 70% ethanol and mix 2min by inversion. Remove 70% ethanol with p1000

• Add 1mL of 10% Domestos plus 0.01% v/v Tween 20 (1µL/mL) for 20min.

• Remove Domestos solution and wash 5X with 1mL sterile water

• Add another 500µL or so of sterile water, mix up the seeds and transfer to MSO plate
by up-ending tubes and flicking. Be careful not to contaminate the seeds by flicking onto
the tube lid – hold tube at an angle.

• Incubate at something like 28°C in the light – no dark or cold treatments necessary.
Germination should occur within 1 week.

Highly useful notes:

• Steps up to the addition of the Domestos solution do not have to be done in the flow.
• If your’e doing lots of lines, use a multi-dispenser pipette for adding the ethanol and the
bleach solutions.
• It is normal for some seeds to sink and some to float. It is not your fault.
• A 30µL volume is about 100 seeds. It’s a good idea to only sterilise volumes less than
250 µL of seeds in this size of tube for ease and efficency.

Nuking with Chlorine gas

• Put the seeds in some kind of container that will not spill the seeds easily and also
allow good contact with the chlorine gas
• Put the containers in a vacuum dome, and that in a fume hood. Place 400mL of 12.5 %
Sodium hypochlorite bleach in a beaker in the dome, add 4ml of conc. HCl, and quickly
put he lid on. The bleach will fizz a bit as a releases chlorine gas. Turn on the vacuum
just enough so that the lid seals, then turn off the vacuum. It’s a good idea to tape down
the lid in case the lid leaks.
• Leave for at least 4-8hrs. 4 is fine, maybe use 8 for really contaminated seed. Much
more time will kill the seed. Alternatively use 7.5% hypochlorite and leave over night.
The seeds will go pale – that’s why they call it bleach
• Sprinkle the seeds onto plates for germination as above. No worries
 58

Tobacco transformation protocol

Before beginning, relax. This is only marginally more difficult than a miniprep and takes
careful misplanning to stuff up.

Triparental mating

The best way to get your plamids into agros. See Agrobacterium Tri-Parental Mating on
p…

The agros
• About 4 days before you want to transform, streak glycerol stock of agro strain
containing plasmid onto a selective YEP plate. This means you can visually check the
quality of your agros.
• Innoculate 200µL of YEP in a 1.5mL tube with a single colony and vortex it. Use this
200µL to innoculate 10mL of selective YEP and grow O.N., shaking fast at 26-28°C.
Small variations in temp are OK, but above 30°C agros can lose their Ti plasmid.
• Next day, about 9am: Measure the A660 of the O.N. cultures – you want cultures that
are about 0.5. Make up dilutions of these cultures in selective YEP to get 10mL at an
A660 of 0.125. Grow these diluted cultures up for another 2-4 hrs to get them to an 660
of 0.4. This theoretically has 1.2 X 109 cells per mL – see PMB Manual B6/6 (1994) for
the theory on this one. Alternatively, trust me and don’t worry about it.

Go transformation

• Cut young leaves into ~7mm2 pieces adaxial side down MSO liquid in a petri dish and
cover with lid. The younger and healthier the leaves are, the better the transformation
efficiency gets.

• Pass the agro culture to a sterile 50mL tube and spin down (leave in shaker until the
last minute) in Jouan at R.T. and 4500 rpm for 10min, remove all YEP and resuspend
by shaking with an equal volume of non selective MSO liquid.

• Pour agro suspension into petri dishes (5mL does one plate and 20 leaf pieces),
transfer tobacco and wound with scalpel by pricking (5-10X per piece). Incubate for up
to 15min. It is important that the internal structure of the leaf does not get drowned or
too damaged.

• Blot on sterile paper.

• Place upside down on non selective MSO plates. Incubate in the dark at 22°C for 3 – 4
days (until some decent bacterial growth is observed).

• Rinse in C500 solution to kill off agros, blot on sterile paper, then transfer to MS9 with
C500 K150 plates. Make sure the pieces have good contact with the media - if they curl
up after about a week, cut them in half.
 59

• Sub every 2 weeks (Kanamycin breaks down in the light and selection is not reliable
after 2 weeks). It is important to keep the plates as free of condensation to reduce
losses of shoots to vitrification. Do this by opening plates and tapping the lid free of
droplets in the flow. You can also leave the plates open for about 5 minutes to dry the
tissue a bit. This little drying routine becomes more necessary as the tissue bulks up
and transpires more.

• Shoots should begin to form after about 3-4 weeks. When they are large enough, cut off
at the base and place individual shoots into universal tubes (use 15mL of rooting media
with C250 K100 for each tube). Take only 1 shoot for each leaf piece to guarantee
distinct lines, and avoid any callus. It is important to include cefotaxime in media up until
transfer to soil because agros are very persistent. It is important to get the shoots off
MS9 and onto rooting media as soon as you can – long exposure to auxin and cytokinin
discourages rooting.

• You can also do a callus growth assay for an additional check of kanamycin resistance
if you want. Take a small (~2mm2 ) piece of leaf tissue from the shoot on rooting media
and put on a gridded callus inducing plate (MS3 C500 K150) and test over a 2 week
period for growth or bleaching. Callus inducing media (MS3): MSO plus 3mg/L NAA and
0.3 mg/L Kinetin.

• Transfer to soil is best done when the shoots have just begun to form decent roots
(about 1–2cm worth of root). This does not have to be sterile, but keep the shoots
covered as much as possible during the whole process to prevent drying. Remove the
plant from the universal tube, wash any agar off the roots in MQ water, then put them in
Quick-pot 30 seedling trays with sterilized UC mix. After transfer, water them with 2g/L
Aquasol + 2g/L Mancozeb. Grow them at 28°C with a 16-hour photoperiod in a growth
cabinet type device until they’re ready to pot. Pot in whatever pot you want, with
sterilized UC potting mix.

• The only real problem after this point is blue mould, a common fungus which can reduce
your healthy plants to dying plants in less than a week. The only effective
preventative/cure is the systemic fungicide Ridomil. Treat your plants according to
manufacturers instructions from transfer to soil onwards. Also note that if your plants are
wilting, it can be because they are over watered.
 60

8.1 Transgenic analysis

Transgenic phenotype analysis for Arabidopsis

For every test:
• All plant growth regulators are filter sterilised and added after autoclaving
• Surface sterilise seeds in an eppendorf:
- Soak in a mixture of 1:1 Ethanol: 3% hydrogen peroxide for 5 mins
- rinse in SDW
• Suspend seeds in sterile 0.15% agar solution for plating
• Use sterile cut yellow tips for dispensing seed
• Seeds should be separated out individually and not allowed to clump together
• For most tests seeds should be plated in two horizontal lines across the plate (only 20-
30 seeds per line)
• Cold treat seeds to syncronise germination - 4°C, dark, 3 days
• Unless otherwise mentioned all plates are to be incubated vertically (held in porcupine
racks) under lights (16 hr light / 8 hr dark cycle)
• Days of growth are counted from the start of incubation under light
• Root and hypocotyl lengths should be measured under the binocular microscope using
the eyepiece micrometer. Make sure you use a consistant magnification (1x is good)
• For every test each line should be grown on plain GM under the same conditions as a
control
• It is a good idea to use both WT and pBI121 controls to compare with your transgenic
results

Germination Medium (GM) for 1L

1x MS Salts 4.31g
1x MS Vitamins 1mL (of 1000x) pH 5.72 with KOH
1% sucrose 10g
1% agar 10g

Plant Growth Regulators

Abscisic Acid (ABA)

Influence on Plant:
- signals to plant it is undergoing physiological stress (ABA content rises under
water, salt and chilling stress)
- induces stomatal closure
- stomatal closure → reduced photosynthesis → reduced growth particularly in
shoots
- inhibits seed germination
- causes root plasma membranes to become more positively charged
- inhibits protein synthesis
 61

Test:
Germinate on plain GM, after 5 days incubation transfer seedlings to 10µM ABA
(only transfer seedlings of approximately the same size), grow for 10 days then
assay root length
Ref: Parcy, F., Giraudat, J. (1997) Interactions between the ABI1 and the
ectopically expressed ABI3 genes in controlling abscisic acid responses in
Arabidopsis vegetative tissues. Plant Journal 11(4): 693-702.

Other tests :
1) Germinate on ABA, assay germination rate
2) Float detached leaves on ABA solution and determine stomatal closure rate

Auxin
Influence on Plant:
- promotes root elongation at low concentration and inhibits at high concentrations
- promotes root initiation
- promotes elongation of detached coleoptiles
- promotes hypocotyl elongation
- stimulates ethylene production

Test:
1) Germinate in dark (wrap plates in alfoil) on 10µM IAA, measure hypocotyl length
after 6 days in dark
2) Germinate on 10µM IAA grow for 10 days then assay root length

Other tests:
Germinate in light on plain GM for 5 days, transfer to 10µM IAA aligning root tips
along a straight line on plate, measure new root growth once a day for six days

Cytokinins (CK)
Influence on Plant:
- promote cell division
- decrease length of S phase in cell cycle (between G2 and mitosis – DNA
synthesis stage)
- promote lateral bud growth
- inhibit primary root growth in light or dark grown seedlings
- retard senescence in leaves and some cut flowers
- enhance growth mainly by water uptake and subsequent cell expansion in
excised etiolated cotyledons
- enhance ability for tissue to act as nutrient sink
- promote leaf growth (slightly)
- enhance chloroplast development in etiolated sedlings (once exposed to light)
- promote grana formation and increase rate of chlorophyll synthesis
- may be intermediate in light response pathways
- downregulate phytochrome production
- ABA inhibits CK metabolism and vice versa
- response most likely mediated by Ca2+ concentration changes
- CK antagonists inhibit CK dependent growth
- most active: substituted pyrollo [2,3-d] pyrimidines and 7-substituted
3methylpyrazolo [4,3-d] pyrimidines (~1µM)
 62

- inhibition can be reversed by increasing CK concentration

- Interaction with ethylene

- produce “triple response” effect on dark grown seedlings – CKs
exaggerate curvature of apical hooks and inhibit hypocotyl elongation
- CKs stimulate ethylene production
- ethylene partly accounts for some CK responses (inhibition of root and
hypocotyl growth)
- ethylene inhibitors (AgNO3, AVG) partially block CK response

Test:
Germinate on 5µM BA, assay root length after 10 days

Other tests:
1) Germinate on 5µM BA, transfer groups of seedlings after 1, 2,3,4 and 5 days
after germination onto plain GM measure root length daily (rate of recovery)
2) Measure root growth over a range of BA concentrations (0.1-10µM)
3) Measure ethylene production over a 24 hr period between day 3 and 4 after
germination
4) Germinate on 5µM BA + 20µM AgNO3 to see if CK inhibition is stopped (see if
response is mediated by ethylene)
5) Germinate on CK antagonists + range of BA concentrations to see if CK
inhibition is stopped (blocking receptor)
6) Transfer etiolated seedlings to CK, assay rate of “greening”
7) Put detached etiolated cotyledons on CK, assay growth
8) Paint leaves with CK solution, examine for reduced senescence

Ethylene
Influence on Plant:
- Triple response (in seedlings) – inhibited stem elongation, increased stem
thickening and horizontal growth habit. Also inhibited leaf expansion and
retarded epicotyl hook opening
- promotes senescence and abscision of leaves
- inhibits flowering (in most species except mango and bromeliads)
- induces flower senescence
- increases percentage of female flowers in dioecious plants
- stimulates fruit ripening

Test:
Incubate on 10µM ACC in dark for 6 days, measure root and hypocotyl lengths

Other Tests:
1) Germinate on 10µM ACC in dark for 3 days, look for triple reponse

Gibberellins
Influence on Plant:

Tests:
Germinate on 10µM GA3, grow for 8 days then measure hypocotyl length
 63

Brassinosteroids
Influence on Plant:
- effects resemble those of auxin
- promote cell elongation and division → growth
- stimulate stem elongation (peduncles in Arabidopsis)
- inhibit root elongation
- induce ethylene production
- compensate for cold and salt stress
- possibly increase tissue sensitivity to auxin but can also act independently of
auxins
- interact with auxin and ABA

Test:
Germinate on 100nM epibrassinolide, assay root length after 10 days
Fusicoccin (FC)
Influence on Plant:
- promotes coleoptile and cotyledon growth
- induces stomatal opening in the dark and reverses ABA inhibition
- promotes seed germination
- activates proton pump causing acidification of external medium

Test:
1) Germinate on 10µM FC, measure hypocotyl length, assay general development
2) Put leaf discs on in buffer with fusicoccin and bromocresol blue indicator (pH),
look for colour change

Jasmonic Acid (JA)

Influence on Plant:
- mediator of stress responses including wounding, water, touch and pathogen
attack
- promotes leaf senescence and abscision
- induces stomatal closure
- promotes chlorophyll degredation (?)
- inhibits root growth
- inhibits seed germination and seedling growth
- inhibits flower bud formation
- induces tuber formation in potato
- induces phytoalexin synthesis

Test:
Germinate on 100nM methyl jasmonate, assay root length

Salicylic Acid (SA)

Influence on Plant:
- induces flowering
- induces heat production in thermogenic species
- inhibits seed germination (?)
- inhibits catalase activity
- induces resistance to pathogens- systemic acquired resistance (SAR)
 64

- production of cell wall components

- phytoalexin production
- enhanced peroxidase activity
- synthesis of proteinase inhibitors
- synthesis of pathogenesis related (PR) proteins
- possibly mediates SAR by binding to catalase leading to increased H2O2
production

Tests:
1) Germinate on SA, determine germination rate (??)

Possible results. Please add pictures of standard or unusual results so that we have a
record of what other people have found.

1. 10µM IAA grown in dark (Naomi, mid-late 1999)

♦ I got a lot of variation in the WT and pBI121 control plants. The antisense plants
grew better.

pBI121 control grown on IAA in dark

WT grown on IAA in dark

More WT grown on IAA in dark

WT grown on GM grew much better.

 65

GUS assays Chris

Pretreatment of Tobacco Tissues
Tissues that were semi-permeable to the GUS histochemical assay mix were
pretreated in cold 90 % acetone for 15-30 min. After acetone pretreatment the tissues
were washed for 5 min in 50 mM Na2HPO4 (pH 7.5) rinse buffer containing 0.5 mM K3Fe
(CN)6 and 0.5 mM K4Fe (CN)6.3H2O (Sieburth et al., 1997).

GUS Histochemical Assays - see also extra section by Dave below

The histochemical assays were performed according to Jefferson (1987) with minor
modifications. Intact seeds, seedlings, plantlets or sections of organs were vaccum
infiltrated for 15-20 min in reaction mixture containing 1 M Na2HPO4 pH 7.0, 0.5 % Triton
X-100, 0.5 M EDTA sodium salt, 0.1 M K3Fe (CN)6, 0.1 M K4Fe (CN)6.3H2O, and 2 mM X-
Gluc (Chemica ALTA, Cananda) dissolved in Dimethyl Sulfoxide (100mM). Infiltrated
tissues were incubated in the GUS assay mixture for 24 hrs at 37 oC or until the blue
indigo dye precipitates. Immediately following the GUS assay the tissues were cleared by
washing in a series of ethanol steps and stored in 60 % ethanol. Tissues were
photogaphed using a dissecting NikonAFX-IIA binocular microscope and Nikon FX-35mm
camera or whole plantlets photographed with a RICOH digital camera, RDC-4300 (DU-4
software).

GUS Fluorometric Assays

Fluorometric GUS assays were based on methods described by Jefferson (1987).
Frozen leaf discs (8-10 ) and plant tissue (100-150 mg) were crushed to a fine powder and
ground for 30 sec at full speed using a Makita drill and micropestle in 450 µL of GUS
extraction buffer (50 mM Na2HPO4 pH 7.5, 10 mM 2-β mercaptoethanol, 10 mM Na2EDTA,
0.1% Sodium Lauryl Sarcosine, 0.1% Triton X-100). Cell debris was pelleted by spinning
for 10 min at 12 000 rpm in a chilled centrifuge. A 300 µL sample was taken from the
clarified extract, protein concetration determined and stored at 4 oC overnight or at –80 oC
for several weeks.
A GUS fluorometric assay mix containing 10 µL of sample extract (2.5 µg of protein)
and 90 µL of GUS assay buffer (GUS extraction buffer + 1 mM MUG) was prepared in a
flat bottom microtitre plate prewarmed to 37 oC. GUS activity was determined by
continuous assays using Labsystems Fluoroscan Ascent FL microfluorometer reader and
 66

software. Successive fluoroesence readings were determined at a wavelength of 365/455

nm over a 40 minute interval. GUS activity was determined from a set of 4-methyl-
unbelliferone standards (supplied by Sigma) and expressed in nmoles 4-MU /min/mg
protein or nmoles 4-MU/min/gfw units.

GUS-plus: useful extra info on GUS histochemical assays – Dave 10/3/00

Staining solution – based on, by slightly different to Sieburth et al., 1997.
Composition Stock solution 20ml
2 mM X-Gluc Dissolve 20mg X-gluc in 0.4ml DMSO 0.4ml
0.5% (v/v) Triton X-100 10% (v/v) 1ml
0.5 mM K3Fe (CN)6, (ferricyanide) 0.1M (3.3g/100ml) 1ml
0.5mM K4Fe (CN)6.3H2O (ferrocyanide) 0.1M (4.2g/100ml) 1ml
10mM Na2EDTA 0.5 M pH 7-8 (14.6g/100ml) 0.4ml
50mM PO4 buffer pH 7.0 1M K or Na PO4 buffer pH 7.0 1ml
water 15.2ml

Other stuff:
• Store X-Gluc at -80°C as powder aliquots
• Ferro and ferri cyanide to be stored in the dark at 4°C. These will probably last year or
more and are fine if they are yellow and have no precipitate
• Chris included Tween 20 at 0.1% (v/v - 20µl per 20ml) in the GUS mix, and apparently
it improves penetration by a small amount
• Chris’ words on acetone pretreatment: “I did use acetone on some tissues but not
others. I don't think Jai or Rebecca used it, since they found it inhibiting the GUS staining,
which it does do in soft/delicate tissues. This is why I only ever used it on seedlings from 8
days to 18 days (tobacco). Anything less then 8 days...acetone inhibited the GUS
reactions. I never used it on tissue culture grown seedlings.......too soft. My advice is to
optimise when and how much time to treat tissues in acetone with arabadopsis CaMV35S
lines. Otherwise cut the tissues up??”
 67

1M Na PO4 buffer pH 7.0

50ml 100ml
IM Na2HPO4 28.85ml 57.7ml
IM NaH2PO4 21.15ml 42.3ml

Adjust pH with a couple of NaOH pellets if necessary

1M Na2HPO4
Using anhydrous Na2HPO4 (F.W. 141.96), 14.2g for 100ml.
Using Na2HPO4.2H2O (F.W. 177.99), 17.79g for 100ml.

1M NaH2PO4
Using NaH2PO4.2H2O (F.W. 156.01), 15.6g for 100ml
 68

Fluorometric GUS assays Gimara

Using the Fluoroskan Fluorometer

Detection of beta-glucuronidase, (GUS) enzyme activity fluorometrically, involves cleavage

of the substrate 4-methyl umbeliferyl glucuronide, (MUG) by GUS relative to that of known
standards with the cleavage product fluorescing at a wavelength distinct form that of
excitation.

Requirements:
− GUS Assay Buffer (GAB)
− GEB
− Microtitre plate (white with replaceable black wells)
− Esky and ice
− Samples stored in the –70°C
− 1M Na2CO3

1. The sample to be assayed should have been already pre-aliquoted and stored at
–70°C.
2. The amount of sample to be loaded is calculated to contain 4µg of protein for each
well. Note that you are going to need 8 representatives of sample for each analysis.

DON’T FORGET +VE GUS CONTROL and –VE CONTROL (Untransformed tissue
treated in the same manner).

BEFORE LOADING THE SAMPLES INTO THE FLUOROMETRIC PLATE MAKE SURE
THAT THE FLUOROMETER HAS BEEN BOOKED & IS ON.

3. Plan a map of how the plate is to be loaded eg:

Samples: 1 2 3 4 5 6 7 8 9 10

10

20

40

60
 69

4.Go to Fluorometer and open up ascent.

− Start
− Programs
− Ascent
− Ascent software for fluoroscan
− Session
− Open
− C:\ascent\gimara (MAKE A COPY OF THIS IN YOUR OWN DIRECTORY, DO NOT
ALTER ANY SESSIONS IN THIS).
− Injab.ses (injects 100µl GAB into wells)
− Check all parameters- ie. the area of template to be dispensed with GAB (located at
bottom of screen, make sure you are on procedure page right side of screen).
− Injectsb.ses (injects 50µl of Stop buffer, NaCO3). Once again check all parameters,
dispense and measure.

5. Leave fluorometer open on Injgab.ses

6. Prime the machine with GUS assay buffer.
− Place the bottle of GUS assay buffer (stored @ 4°C in alfoil) and place inside
fluorometer)
− The end of the black plastic tubing with the white cap is place into the GAB bottle.
− The other end of the black plastic tubing with the probe is placed into a waste
beaker.
− Execute
− Prime, Click on 1.
7. Place the prove into hole M (make sure in Lower position see lid of fluoroskan for
instructions).
8. Load samples into microtitre plate (white tray with replaceable black wells).
9. Load plate into fluorometer. (Cut corner left hand foremost is the correct orientation for
well 1).
10. Start
(100µl of GAB should now be loaded into each well).
11. Once this is down. Remove the GAB. Empty the probe, (EXCUTE, EMPTY, 1).
12. Replace the GUS bottle with the Stop Buffer.
13. Prime in the same manner as before, then put probe into M.
14. Open injectsb.ses
15. Start
16. Results can be found on Results page (see right hand side of screen for tab save onto
Excel sheet do not clog up Ascent directory with results).

DON’T FORGET TO CLEAN UP FLUOROSCAN.

Standards:
Standards are made for 1mM Methylumbelliferone Stock solution (lasts only 1 month).. A
range of stardards from 125pm/well to 4000pm/well are prepared from GEB and 1M
Na2CO3. Eg:
500µl of 40µM 4MU +500µl GEB (0.02M or 20µM 4Mu) 100µl of this +50µl 1M
Na2CO3 per well
= 4000pm.
 70

Calculations:
1. Mean of 2 RFUs for standards & unknowns.
2. Generate standard curve, RFUs vs picomoles/well slope +correlation. (disregard 1st
time point as not linear)
3. This change in RFU/minute is for 4µg protein delta RGU/min/mg protein.
4. Using calculated slope from Step 2 apply to unknowns to give pm4MU/min/mg.

Solutions:
GEB (100ml)
0.5M Na2HPO4, pH7 10ml
1M DTT 1ml
0.5M Na2EDTA, pH8 200µl
10% Sarcosyl 1ml
5% Triton X-100 2ml
H2O 86.8ml

GAB
1M 21.14mg/60ml GEB (Stored in dark, 4°C for a few weeks).

STOP BUFFER
21.2g Na2CO3 in 1L of H2O

1mM Methylumbelliferone Stock Solution

19.82mg in 100ml (H2O) (Stored in dark, 4°C for 1month).
 71

9. Other Procedures

Exonuclease III Digests; (Sequencing using deletions)

Tony

In pBluescript use these enzymes:

M13 end: use KpnI (3’ overhang) and HindIII (5’ overhang)
rM13 end: use SacI (3’ overhang) and BamHI (5’ overhang)
In pGEM-T use these enzymes:
M13 end: use AatII (3’ overhang) and NcoI (5’ overhang)
rM13 end: use SacI (3’ overhang) and NotI (5’ overhang)

The 5’ overhang allows Exonuclease III (ExoIII) to cut into the plasmid, while the 3’
overhang protects ExoIII from digesting in the other direction.

Cut DNA:
approx. 10µg DNA
5 units of each enzyme
1µL of RNase
Add correct buffer
Add ddH2O to 20µL

Extract with phenol-SEVAG and ethanol precipitate the DNA, resuspend the DNA in 80µL
ExoIII buffer, run 5µL on a gel to ensure full digestion of the DNA

Set up three tubes

Tube Volume 0.2M NaCl Time Points (min) (can be changed for
larger insert size)
1 67.5 1, 4, 8
2 67.5 10, 15, 20
3 90 25, 30, 35, 40

Place tubes on ice.

Add 360units of ExoIII to DNA and incubate at 30°C. At the time points indicated above
remove 7.5µL into the corresponding tubes. As each tube is completed place tubes at
70°C for 10min, then on ice. Run 5µL of each tube on a gel.

Add 20µL Mung Bean Buffer, add 1unit of Mung Bean Nuclease and incubate 30°C for
30min.
To make the dilution of the Mung Bean Nuclease:
Take 1.1µL Mung Bean Nuclease (10u/µL) and add to 2.2µL Mung
Bean Buffer, use 1µL per tube.

Stop the reaction by adding 6µL of 0.5M Tris - 0.125M EDTA. Add 30µL to tubes 1 and 2.
Run 6µL on a gel.
 72

Phenol SEVAG and ethanol PPT the DNA, add 14µL Na acetate and 280µL ethanol,
resuspend the DNA in 10µL 20mM Tris- 7mM MgCl2.

Run 1µL on a gel to ensure ranges of sizes.

Add 1µL of Klenow to the remaining DNA; incubate 37°C for 2min
Add 1µL dNTPs; incubate for a further 2min

Ligate:
11µL DNA
2µL Buffer
2µL T4 Ligase (0.5units/µL)
5µL ddH2O
Incubate 15°C overnight

Phage hints
• Phage should NEVER be left out at RT and NEVER be frozen at -20°C or -80°C; phage
should ALWAYS be kept at 4°C during storage or on ice during handling.

• Always mix phage well before each operation – they occur as a suspension, not a
solution, and thus settle quite quickly after mixing.

• The 20min incubation of bacteria + phage is to allow the phage to infect the bacteria
via the maltose transporter (receptor), the production of which is induced by growing
the bacteria in maltose. If you are in a hurry, this time can be reduced to 12-15min with
little effect on efficiency of infection.

• When transferring the LB + 0.7% agarose + 10mM MgSO4 to the bacteria after the
20min incubation, use 5mL pipette tips prewarmed in the drying oven, and dispense
the liquid against the inside of the 15mL tube to cool it slightly and reduce bubble
formation.

• LB plates for library screening should be incubated inverted at 37°C for several hours
prior to plating so that they are: (a) warm enough to give you time to spread the
agarose evenly over the top before it melts, and (b) dry enough so that condensation
doesn’t form post-plating and lead to streaking or excessive diffusion of plaques.

• Plaques are visible as clear areas on a ‘lawn’ of confluent bacterial colonies which
grow on the surface of the top agarose layer. Sometimes it is difficult to tell if you have
confluent lysis on a plate or simply an intact bacterial lawn representing no phage
infections. In such cases, it is best to show it to someone else to get their opinion, sniff
it (does it smell of bacs?) or re-plate at a range of dilution (more and less
concentrated).

• Pre-cooling of plates prior to taking plaque lifts prevents smearing of the colonies and
separation of the top agar layer.
 73

• Don’t throw out your hybridisation probe solution from your primary screening – store it
in a labelled 50mL tube at RT in the Radiation Lab until you need it for secondary
screening. Boil the entire tube for 10-15min, quench on ice, and use it to replace the
prehybridisation solution.

cDNA Library Screening Andrew 07/11/99

Note that this procedure was developed on libraries in the lambda ZAP vector

Preparing plating bacteria

• Inoculate 10mL LB + 0.2% maltose + 10mM MgSO4 with a single large E. coli XL1-Blue
colony
• Incubate overnight at 37°C with shaking (>8 hrs)
• Add a 3mL aliquot of the overnight culture to 3mL of sterile SM, pH 7.5
• The diluted cells can be stored for up to 2 weeks at 4°C, but fresh is best

Plating phage for library screening

• Plate onto 150mm plates for primary library screening (10,000 to 50,000 pfu per plate)
and onto 90mm plates for titrations and secondary library screening (20-200 pfu per
plate)

• Add 1-10µL of phage suspension (calculated from known titre to give an appropriate
number of pfu per plate) to 600µL (150mm plates) or 200µL (90mm plates) of the
diluted plating bacteria in a sterile 15mL tube and mix well – you should have one tube
for each plate you plan to use
• Incubate tubes in a 37°C waterbath for 20min
• Add 10mL (150mm plates) or 3.3mL (90mm plates) LB + 0.7% agarose + 10mM
MgSO4 (melted in microwave and kept at 50°C) to each tube
• Roll the tube vigorously between your hands for several seconds to mix, and pour
quickly onto a warmed 1.5% agar LB plate
• Quickly swirl the molten agarose on the suface of the plate to give even coverage of
the surface and allow it to set on a level suface for a few minutes
• Incubate the plates inverted at 37°C for 9-14hrs

Plaque lifts to nylon filters

• Pre-cool the plates containing the plaques you want to screen at 4°C for at least 30min
prior to taking the lifts

• Take a labelled nylon filter of the correct size and hold it (gloves on!) such that it bends
in the middle like a trough
• Carefully lower the trough onto the surface of the plate and allow it to wet whilst holding
it there – once the filter is in contact with the agar, DO NOT MOVE IT AGAIN
• Release the filter and allow the sides to fall onto the suface of the plate as the
moistures seeps through
• Allow the filter to sit on the surface of the plate for 6min
• During this time, use a fine needle to make an asymmetric pattern of orientation marks
through the filter and into the agar below
• Lift the filter off the plate with one quick, even movement using a pair of filter forceps
• Place the filter phage side up on a piece of 3MM Whatman paper soaked (no free
liquid) in denaturation solution for 6min, then neutralisation solution for 6min, then a
 74

vigourous wash in 2x SSC and put on a filter paper to dry (denaturation and
neutralisation solutions are same as for Southern blotting)
• UV crosslink the phage DNA to the filter using the pre-programmed Southern filter
protocol
• For each plate, a duplicate filter is prepared by repeating the above procedure but
incubating the filter on the plate for 12min, and using the orientation marks for the first
filter to mark the second

Probing library filters and identifying positives

• Prehybridise the crosslinked library filters at 65°C in Church buffer as per the standard
Southern protocol, and make a probe using the DNA fragment you want to screen
against the library
• Hybridise the probe (diluted in prehybridisation solution to about 0.5 million cpm/mL)
overnight at 42°C
• Wash the filters at the appropriate stringency for your purposes (homologous probes at
high stringency, heterologous probes at low stringency)
• For primary screening, seal the washed filters in plastic as duplicate pairs and lay them
to phosphor screens; print the image at full size and align image to filters using the filter
outlines (mark alignment holes from plates onto images)
• For secondary screening, dry filters, magic-tape down to a piece of stiff card, staple to
a sheet of X-ray film and expose overnight at -80° with two intensifying screens; align
developed film to filters using the staple holes (mark alignment holes from plates onto
autorads)
• Compare the original and duplicate library filter images; circle the ‘true’ positives
• Align the images containing the circled ‘true’ positives with the respective library
screening plate using alignment holes on plates and corresponding marks on images;
mark in the position of the plaque or region corresponding to the positive signal

Isolating primary positives and secondary screening

• Place 500µL of SM buffer, pH 7.5 in an Eppendorf tube for each positive plaque
identified
• Carefully break the thin end off a disposable glass pastuer pipette and place a pipette
bulb over the broken end (use a fresh pipette for each plaque)
• Expel the air form the bulb and line up the unbroken end of the pipette with the chosen
positive plaque
• Using a rapid motion, stab down through the agar to the bottom of the plate and
release the suction on the bulb
• The agar plug can be retrieved by wiggling the pipette from side to side, and ejected
into the labelled tubes containing SM
• Allow the tubes to stand at RT for 1-2hr (or overnight at 4°C) to allow the phage to
diffuse out into the SM
7 8
• The titre of this phage soak stock should be ~10 to ~10 pfu in 500µL; infect bacteria
with 1µL of a 1:100 dilution (1µL phage soak plus 99µL SM), plate on 90mm plates,
and lift and probe as described above
 75

Isolating secondary positives and in vivo excision of pBS-cDNA

• Place 200µL of SM buffer, pH 7.5 in an Eppendorf tube for each secondary positive
plate
• Touch the end of a sterile toothpick to the centre of a well-isolated positive plaque and
agitate it vigorously in the SM buffer for several seconds to dislodge the adherent
phage

• Add 50µL of your secondary positive plaque to 50µL of an undiluted overnight culture
of XL1-Blue cells in a 15mL tube, mix well, and then add 2µL of the stock ExAssist
helper phage
• Incubate in a 37°C waterbath for 15min, add 3mL LB, and incubate for 3hr at 37°C with
shaking
• At the end of the 3hr incubation, spin the 15mL tubes for 15min at 2000rpm
• Decant the supernatant into fresh 15ml tubes, heat to 70°C for 15min, and spin for
15min at 4000rpm
• Add 100µL of the supernatant (= excised infectious phagemid) to 150µL of an undiluted
overnight culture of SOLR cells in an Eppendorf tube – keep the remaining supernatant
at 4°C just in case
• Incubate in a 37°C waterbath for 15min, plate 100µL on LB + 100µg/mL ampicillin, and
incubate overnight at 37°C

Tertiary screening and sequencing of your positive cDNAs

• Tertiary screening by PCR is easiest if you are looking for, say, the full-length version
of a cDNA for which you already have a partial clone
• This process simply involves colony PCR on several replicate colonies (at least two)
from your in vivo excision plates. Amplify using (a) the T7 and T3 primers for 35 cycles
of 50°C annealing and a generous extension time to tell you the total size of the cDNA
insert, and (b) either of these primers and a sequence-specific primer facing in the
opposite direction to check that you picked phage from a positive plaque in the
secondary screen. Do 25µL reactions and run 10-20µL out on a 0.8% agarose gel with
suitable markers (lambda H/E should be sufficient)
• For tertiary screening by Southern blotting and hybridisation, see Maniatis et al.
• Miniprep, PEG precipitate and sequence your positive plasmids as per usual – use the
T3 primer for good 5’ end sequence from the Weigel flower cDNA library (EcoRI-XhoI
directionally cloned)
 76

Electrocompetent cells
For 500mL culture prepare:

2X autoclaved GSA tubes

1L sterile ddH2O
at least 50mL sterile 10%(v/v) glycerol
adequate microcentrifuge tubes for aliquots of cells (500mL makes ~70 tubes)

Method N.B: Keep solutions and equipment cold and sterile

- A single colony of DH10B cells is used to inoculate a 5mL LB starter culture, incubate
at 37°C overnight.
- Inoculate 500mL LB with the starter culture, incubate at 37°C, take spectrophotometric
reading periodically at 600nm
- Grow culture until it reaches an OD(600nm) above 0.500. This usually take about 2.5
hours, but may take longer.
- Chill the flask for 30min on ice.
- Centrifuge cells in 2XGSA tubes, at 5000rpm/15min/4°C
- Discard supernatant and resuspend cells in 250mL ddH2O each tube.
- Recentrifuge GSA tubes at 5000rpm/15min/4°C
- Discard supernatant and resuspend cells in 250mL ddH2O each tube.
- Recentrifuge GSA tubes at 5000rpm/15min/4°C
- Discard supernatant and resuspend cells in 20mL 10%(v/v) glycerol each tube.
Transfer into a 50mL tube
- Centrifuge cells in the Jouan centrifuge at 3000rpm/15min/4°C
- Discard supernatant and resuspend in 3mL10%(v/v) glycerol.
- Aliquot 42µL into eppendorf tubes and snap freeze in liquid nitrogen
- Store at -80°C

To test the cells

Electroporate new cells and a tube of old cells (as control) with 1µL pBS (5ng/µL), plate
out 100µL onto an LB-amp plate.
Cell density on the plates should be confluent (or very close too).
 77

in situ Hybridisation Dave

I. Tissue fixing, embedding and sectioning

A. Fixation

50% FAA solution

Ethanol @100% 50 45
Glacial acetic acid 5 5
Formaldehyde @37% 10 5
MQ water 35 45
________________
100 100

• Immerse tissue in 50%FAA solution

• Infiltrate under vacuum 15min. Pull the vacuum slowly. Release the vacuum and
repeat. The tissue should sink after the second step.

• Incubate without vac 2hrs RT

• Vac infiltrate another 15min

• Incubate without vac 1hr.

B. Dehydration

• Decant off fixative and replace with 50% ethanol and incubate RT 30min. Repeat this
step.

• Replace 50% with 60% and incubate 30min

• Repeat for the following solutions: 70, 85, 95% ethanol. leave ON in 95%.

• Replace 95% with 100% and incubate 1hr, then repeat for 30min

C. Clearing

• Decant 100% and replace with 25%xylene:75% ethanol and incubate RT 30min

• Repeat for the following xylene:ethanol solutions: 50:50, 75:25

• Decant 75% xylenes and replace with 100% xylene and incubate RT 1hr. Repeat this
step twice
 78

D. Infiltration – see John ‘Pauncho’ Bertram for this.

E. Sectioning – see John ‘Pauncho’ Bertram for this.

II. Making DIG labelled probes

A. Transcription (see also Boehringer Applications Manual, p44)

Set up transcription reaction using Boehringer kit:

10X transcription buffer 2.0

10X NTP mix 2.0
RNase inhibitor 1
template x (1µg)
RNA polymerase 2
DEPC water to 20

2hrs @ 37°C. Longer times do not increase yield.

B. DNasing

Add 2µL DNase and incubate 37°C 15min. Stop with 1µL RNase free 0.5M EDTA, with or
without DNase treatment.

C. Clean-up

Add 2.5µL 4M LiCl and 75µL chilled ethanol, precipitate at least 30 min at -80°C or 2hrs at
-20°C. Spin down 15min, wash pellet with 50µL chilled 70% ethanol. Vac dry briefly and
dissolve in 50µL DEPC water.

D. Probe Hydrolysis – for those with time to burn.

(taken from Boehringer “Non Radioactive In Situ Hybridisation Application Manual” p142)

to 50µL labelled probe from above, add 30µL 0.2M Na2CO3 and 20µL 200mM NaHCO3.
Hydrolyse at 60°C for a time determined by the equation:

t=(L0-Lf) / (K*L0*Lf)

where L0 = starting length of probe RNA in kb

Lf = length of required probe RNA in kb (0.2 kb is a good size)
K = rate constant (in this case 0.11kb/min)
t = hydrolysis time in min
 79

Purify the hydrolysed probe by adding:

5µL 10% acetic acid

11 µL 3M NaAc pH 6.0
1µL tRNA @10mg/mL
1.2 µL 1M MgCl2
300µL ethanol ice cold

Ppt @ -80°C 45min, spin 13K 4°C 30 min. Discard supernatant and vac dry (no 80%
ethanol wash). Store @-80°C.

E. Checking probe

This method is rough at best and a better option is to use Boehringer DIG quantification
test strips. Firstly dilute an aliquot of the precipitated probe (50µL) 1:10. Spot 1µL of the
1:10 dilution and Boehringer standards onto a piece of hybond, let dry, then UV cross link.
A useful set of standards to use is 25, 10, 5, 1 ng plus 500 and 100pg. Make these
standard solutions so that 1µL is spotted as for the test. Then treat the membrane:

• Wet buffer 5 min

• Blocker buffer 30min

• Wash in Wet buffer

• 10mL Wet buffer + 10 µL Boehringer anti DIG antibody 30min

• Wash 2X15 min in Wet buffer

• Wash briefly in no PVA colour buffer – colour substrates

• Develop in 10mL no PVA colour buffer

III. Hybridisation

A. Removing the wax

• Xylene inactivates RNases so xylene jars do not need to be baked. Keep xylene in
fume hood.

• Place slide rack in 1st jar of xylene10min. Transfer to 2nd jar and repeat. The same 2
lots of xylene can be used to process many racks.

• Transfer to 100% AR ethanol and dip up and down 15 times or until streaks disappear.
Repeat with the 2nd jar of 100% AR ethanol. Process the slides through the following
ethanol solutions: 95, 85, 70, 50, 30, 15, MQ, MQ. Dip up and down 15 times or until
streaks disappear as before. Change the last MQ for each rack of slides. Save ethanol
solutions.
 80

Making ethanol solutions for 500mL volume:

% ethanol solution mL water mL ethanol

95 25 475
85 75 425
70 150 350
50 250 250
30 350 150
15 425 75

C. Acetylation Reaction

This reaction acetylates any remaining positive charges on the tissue or slides to reduce
background. The half life of acetic anhydride in an aqueous solution is less than 1 min.

• For each slide rack, weigh out 3g of triethanolamine into a small glass jar and dissolve
in 200mL of MQ to make a 100mM solution. Add 1mL conc. HCl, stir and check pH with
test strip - should be 8.0.

• Set up jar with 200mL triethanolamine buffer with stirrer bar stirring hard, add 0.5mL
acetic anhydride, allow to mix briefly, turn off stirrer bar and add slide rack. Leave for 5
min.

D. Dehydration

• Wash the slides in jar of 2XSSC

• Process slides through the following ethanol solutions (use solutions from step C
except 100 and 95): 15,30, 50, 70, 85, 95, 100.

• Vac dry slides (takes ~1hr) Can rank slides according to section quality at this point if
you are useless at sectioning.
 81

E. Hybridisation

Hyb solution:
1X 5X 5mL

50% formamide 62.5µL 312.5 2.5mL

1X Hyb salts @10X 12.5 62.5 0.5
1X Denhardt’s solution @50X 2.5 12.5 0.1
10% dextran sulphate @50% 25 125 1.0
70mM DTT @1M 8.75 43.75 0.35
150 µg/mL tRNA @10mg/mL 1.875 7.5 0.075
500 ng/mL RNA DIG probe @5ng/µL 6.25 (31.25) (0.25)
DEPC water 6 30 0.225

TOTAL(µL) 125 625 5000

• For 5mL premix minus probe, aliquot 118.75µL + 6.25µL probe @5ng/µL for 1 slide (or
593.75µL + 31.25µL probe @5ng/µL for 5 slides.

• Note: 125µL does one slide with about 4 sections, depending on how large each
section is. Include 500 µg/mL PolyA if hybridising with cDNA clone.

• How to do it: Boil and chill probe aliquot in tube, then add rest of hyb mix to
this(premixed in another tube and heated to 42°C). Pre-warmed hyb soln is easier to
spread. Apply to the sections without introducing bubbles, making sure it completely
covers them. Place slides in ninc dishes on cut Kimwipes soaked with ~2mL humidifier
solution with 2 glass rollers on top to elevate slides. Incubate ON at 42°C. Seal dishes
with parafilm and wrap with aluminium foil.

E. Washes and RNase treatment

• Place slides into racks and soak in 3 changes of 4XSSC, about 5 min each. Take care
not to put slides in the same slot in the slide holder because this will damage the sections.
Dip up and down.

• Preheat 200mL RNase buffer in a glass staining jar to 37°C - test temperature with a
thermometer. Add 25µL of 10mg/mL RNase stock to make a 1µg/mL solution. It is a good
idea to use glassware for RNase work so that you can bake it to prevent large scale lab
RNase contamination.

• Place slides in and incubatefor 30min at 37°C

• Wash slides in 3 changes of 2XSSC, then do 2X 20min washes in 2XSSC at RT.

Preheat staining dishes with standard volume of 200mL wash for about 30 min.

• Wash briefly in 3 changes of 1XPBS. Can leave in the last PBS O.N., otherwise
proceed to detection
 82

F. Detection

At this point, you can throw away all your slides and never find out what signal you got.
OR, you could do the colour detection. All steps on rocker in shallow lunchboxes with lids
on unless noted. 40 mL of solution needed for each box which takes 7 slides. Briefly drain
slides on tissue between solutions. Each solution to be put in new box. Take care when
transferring slides and during incubation that slides do not overlap because it is easy to
damage sections.

• Wet slides in wet buffer 5 min

• Block in blocker buffer 1 hr

• Wash buffer 1hr

• Antibody buffer 1 ½ hrs. Use about 15mL of antibody buffer to save antibody. Make up
antibody buffer in one lot in a new 50mL tube to ensure consistency. Do not save the used
solution.

• Wash buffer 4 X 20 min

• Wet buffer 5 min

• No PVA Colour detection buffer 5min

• PVA colour detection buffer in ninc dishes. Wrap in alfoil with lids on – no seal needed
– and leave in 30°C incubator O.N., not on the rocker. Can incubate longer, but most of
the colour is present after an over night incubation.

• Stop reaction when you’re satisfied by washing slides in 2 changes of MQ water, using
slide racks and glass jars.

• Mount using Omnimount medium when slides are dry.

G. tgb1 in situ conditions

• 0.5U/mL anti-DIG antibody 11/2 hrs

• 250ng/µL probe
• 42°C hyb
• RT 2XSSC washes (can do 50 - 55°C but does not reduce background and only
increases damage to sections)
• No proteinase K treatment
• 1µg/mL 30 min RNase treatment
• No coverslips in any step
 83

H. Solutions

For those who are paranoid, I’ve checked all calculations.

1M Dithiothreitol F.W. 154.2

1.542g of RNase free grade DTT in 10mL DEPC water

5M NaCl

73.05g in 250mL. DEPC treat.

1M Tris pH 7.5

30.285g in 250mL. Make up RNase free – do not DEPC treat

Humidifier solution
50mL
300mM NaCl 3mL @5M
50% Formamide 25mL
MQ water 22mL

50% Dextran sulphate

Measure out 10mL in a new 15mL tube with DEPC water, remove water, then add dextran
sulphate and add water gradually to mark.

10X Hyb Salts

10mL – use RNase free solutions
3M NaCl 6mL @5M
100mM Tris pH 7.5 1mL @1M
10mM EDTA pH 8.0 200µL @0.5M
DEPC water 2.8mL

20XSSC RNase free

2L 1L 250mL
NaCl 350.6g 175.3g 43.83g
NaCitrate 176.4g 88.2g 22.05g

10X PBS stock

1L 500mL
NaCl 80g 40g
KCl 2g 1g
Na2HPO4 14.4g 7.2g
KH2PO4 2.4g 1.2g

pH to 7.4
 84

RNase Buffer
1L 500mL
10mM Tris 1.2114g 0.6057g
500mM NaCl 29.22g 14.61g
1mM EDTA 2mL 1mL @0.5M

pH to 7.5

Wet buffer
2L 1L
100mM Tris 24.228g 12.114g
150mM NaCl 17.532g 8.766g
pH 7.5

Blocker buffer

Wet buffer plus 0.5%Boehringer blocking reagent (0.75g /150mL). Add wet buffer to
weighed blocking reagent and microwave/stir carefully until dissolved.

Washer buffer

Wet buffer plus 1% BSA (1g/100mL; 7g/700mL) and 0.3%Triton X-100 (3mL/100mL at
10%; 21mL/700mL).

Antibody buffer

Washer buffer plus anti DIG antibody to 0.5U/mL or 1:1500 dilution (0.67µL/mL at
0.75U/µL; for 50mL use 33.5µL).

No PVA Colour buffer

1L 500mL
100mM Tris HCl 12.114 6.057g
100mM NaCl 5.844 2.922g
50mM MgCl2 50mL 25mL @1M

pH to 9.5, add NBT and BCIP fresh before use.

 85

PVA Colour detection buffer

Make Tris/NaCl solution first, pH to 9.0, heat to 90°C, add PVA and dissolve, then cool to
RT. It Is important to stir very well while heating, or PVA will form hard crust on bottom of
beaker. Check final solution for clarity because PVA tends to form small insoluable flakes.
If necessary, filter through 2 layers of miracloth.

500mL
10% 70-100 KDa PVA 50g
100mM Tris 6.057g
100mM NaCl 2.922g

Add to stock solution before use:

50mL
5mM MgCl2 5µL/mL @1M 250µL
0.2mM NBT see below 165µL
0.2mM BCIP see below 75µL

NBT F.W. 817.6

Boehringer kit: (for 76.92mg/mL or 94.083998mM)100mg in 1.3mL 70% DMF (910µL DMF
+ 390µL water), use at 2.2µL/mL (2.12576µL exactly)

For Promega pre-made 50mg/mL stock in 8% methanol, use at 3.3µL/mL (3.2704

exactly); 165µL/50mL

BCIP F.W. 370.4

Boehringer and Promega BCIP: (for 50mg/mL or 134.9892mM) 50mg in 1mL DMF. Use at
1.5µL/mL (1.4816µL exactly); 75µL/50mL

1M MgCl2 (MgCl2.6H2O F.W. 203.3)

10.165g in 50mL
50.825 in 250mL

Acid Alcohol for cleaning slides and racks prior to baking

2L 1L
Wash grade ethanol 1400mL 700mL
Conc HCl 64mL 32mL @32%
 86

10. Appendixes

Appendix A: Restriction Digests Buffer Tables and

Recognition Sequences
Gibco BRL buffers 1-4 are in a 10x concentration, use 1µL per 10µL total reaction
volume
OPA buffers are also 10x concentration, but use amount specified
0.5x, use 0.5µL per 10µL reaction
1x, use 1µL per 10µL reaction
2x, use 2µL per 10µL reaction
NEB buffers 1-4 are in a 10x concentration, use 1µL per 10µL total reaction volume
When using 2 enzymes, use the buffer system that allows both enzymes to have the
same buffer.
 87
Bbv I GCAGC(8/12)| BstF5 I GGATGNN|
Restriction enzyme BbvC I CC|TCAGC BstN I CC|WGG
regonition sites: BceA I
Bcg I
ACGGC(12/14)|
|(10/12)CGANNNNNNTGC(12/10)|
BstU I
BstX I
CG|CG
CCANNNNN|NTGG
BciV I GTATCC(6/5)| BstY I R|GATCY
R = G or A Bcl I T|GATCA BstZ17 I GTA|TAC
K = G or T Bfa I C|TAG Bsu36 I CC|TNAGG
B = not A (C or G or T) BfrB I ATG|CAT Btg I C|CPuPyGG
V = not T (A or C or G) Bgl I GCCNNNN|NGGC Btr I CAC|GTC
Y = C or T Bgl II A|GATCT Bts I GCAGTGNN|
S = G or C Blp I GC|TNAGC Cac8 I GCN|NGC
D = not C (A or G or T) Bmr I ACTGGG(5/4)| Cla I AT|CGAT
N = A or C or G or T Bpm I CTGGAG(16/14)| Dde I C|TNAG
M = A or C BsaA I YAC|GTR Dpn I GA|TC
W = A or T BsaB I GATNN|NNATC Dpn II |GATC
H = not G (A or C or T) BsaH I GR|CGYC Dra I TTT|AAA
Numbers like: (1/4)| means 1 N on the Bsa I GGTCTC(1/5)| Dra III CACNNN|GTG
top strand, 4 N on the bottom strand BsaJ I C|CNNGG Drd I GACNNNN|NNGTC
then a cut site BsaW I W|CCGGW Eae I Y|GGCCR
BsaX I |(9/12)ACNNNNNCTCC(10/9)| Eag I C|GGCCG
Aat II GACGT|C BseR I GAGGAG(10/8)| Ear I CTCTTC(1/4)|
Acc65 I G|GTACC Bsg I GTGCAG(16/14)| Eci I GGCGGA(11/9)|
Acc I GT|MKAC BsiE I CGRY|CG EcoN I CCTNN|NNNAGG
Aci I C|CGC BsiHKA I GWGCW|C EcoO109 I RG|GNCCY
Acl I AA|CGTT BsiW I C|GTACG EcoR I G|AATTC
Afe I AGC|GCT Bsl I CCNNNNN|NNGG EcoR V GAT|ATC
Afl II C|TTAAG BsmA I GTCTC(1/5)| Fau I CCCGC(4/6)|
Afl III A|CRYGT BsmB I CGTCTC(1/5)| Fnu4H I GC|NGC
Age I A|CCGGT BsmF I GGGAC(10/14)| Fok I GGATG(9/13)|
Ahd I GACNNN|NNGTC Bsm I GAATGCN| Fse I GGCCGG|CC
Alu I AG|CT BsoB I C|YCGRG Fsp I TGC|GCA
Alw I GGATC(4/5)| Bsp1286 I GDGCH|C Hae II RGCGC|Y
AlwN I CAGNNN|CTG BspD I AT|CGAT Hae III GG|CC
Apa I GGGCC|C BspE I T|CCGGA Hga I GACGC(5/10)|
ApaL I G|TGCAC BspH I T|CATGA Hha I GCG|C
Apo I R|AATTY BspM I ACCTGC(4/8)| Hinc II GTY|RAC
Asc I GG|CGCGCC BsrB I CCG|CTC Hind III A|AGCTT
Ase I AT|TAAT BsrD I GCAATGNN| Hinf I G|ANTC
Ava I C|YCGRG BsrF I RCCGG|Y HinP1 I G|CGC
Ava II G|GWCC BsrG I T|GTACA Hpa I GTT|AAC
Avr II C|CTAGG Bsr I ACTGGN| Hpa II C|CGG
Bae I |(10/15)AC(N4)GTAPyC(12/7)| BssH II G|CGCGC Hpy188 I TCN|GA
BamH I G|GATCC BssK I |CCNGG Hpy188 III TCNNGA
Ban I G|GYRCC BssS I C|ACGAG Hpy99 I ACNGT|
Ban II GRGCY|C BstAP I GCANNNN|NTGC HpyCH4III ACN|GT
Bbs I GAAGAC(2/6)| BstB I TT|CGAA HpyCH4IV A|CGT
BstE II G|GTNACC HpyCH4V TG|CA
 88
Hph I GGTGA(8/7)| Sac II CCGC|GG
Kas I G|GCGCC Sal I G|TCGAC
Kpn I GGTAC|C Sap I GCTCTTC(1/4)|
Mbo I |GATC Sau3A I |GATC
Mbo II GAAGA(8/7)| Sau96 I G|GNCC
Mfe I C|AATTG Sbf I CCTGCA|GG
Mlu I A|CGCGT Sca I AGT|ACT
Mly I GAGTC(5/5)| ScrF I CC|NGG
Mnl I CCTC(7/6)| SexA I A|CCWGGT
Msc I TGG|CCA SfaN I GCATC(5/9)|
Mse I T|TAA Sfc I C|TRYAG
Msl I CAYNN|NNRTG Sfi I GGCCNNNN|NGGCC
MspA1 I CMG|CKG Sfo I GGC|GCC
Msp I C|CGG SgrA I CR|CCGGYG
Mwo I GCNNNNN|NNGC Sma I CCC|GGG
Nae I GCC|GGC Sml I C|TYRAG
Nar I GG|CGCC SnaB I TAC|GTA
Nci I CC|SGG Spe I A|CTAGT
Nco I C|CATGG Sph I GCATG|C
Nde I CA|TATG Ssp I AAT|ATT
NgoM IV G|CCGGC Stu I AGG|CCT
Nhe I G|CTAGC Sty I C|CWWGG
Nla III CATG| Swa I ATTT|AAAT
Nla IV GGN|NCC Taq I T|CGA
Not I GC|GGCCGC Tfi I G|AWTC
Nru I TCG|CGA Tli I C|TCGAG
Nsi I ATGCA|T Tse I G|CWGC
Nsp I RCATG|Y Tsp45 I |GTSAC
Pac I TTAAT|TAA Tsp509 I |AATT
PaeR7 I C|TCGAG TspR I NNCAGTGNN|
Pci I A|CATGT Tth111 I GACN|NNGTC
PflF I GACN|NNGTC Xba I T|CTAGA
PflM I CCANNNN|NTGG Xcm I CCANNNNN|NNNNTGG
Ple I GAGTC(4/5)| Xho I C|TCGAG
Pme I GTTT|AAAC Xma I C|CCGGG
Pml I CAC|GTG Xmn I GAANN|NNTTC
PpuM I RG|GWCCY
PshA I GACNN|NNGTC
Psi I TTA|TAA
PspG I |CCWGG
PspOM I G|GGCCC
Pst I CTGCA|G
Pvu I CGAT|CG
Pvu II CAG|CTG
Rsa I GT|AC
Rsr II CG|GWCCG
Sac I GAGCT|C
 89

Appendix B: Solutions
SOB: SOC:
20g Peptone 10mL SOB
5g Yeast extract 100µL 2M Sterile Glucose
10mL 1M NaCl
1mL 2.5M KCl
Make volume up to 1L with ddH2O and Autoclave
Add 10mL 2M Mg+ Solution (1M MgSO4 and 1M MgCl2) to 1L SOB

LB-Amp Plates: 3M Sodium acetate pH5.2:

Dissolve 12.5g of LB in 400mL of ddH2O 49.2g sodium acetate in 160mL ddH2O
Make volume up to 500mL with ddH2O Adjust pH to 5.2 with glacial acetic acid
Transfer into a 1000mL erlenmyer flask and Make volume up to 200mL with ddH2O
add 7.5g of agar and Autoclave
Allow agar to cool to 55°C
Add 1000µL 50mg/mL Amp, Swirl gently and pour plates

Solution 1: Miniprep Solution 2: Miniprep

1.8g Glucose 1.6g NaOH
4mL 0.5M EDTA 20mL 10% SDS
5mL 1M Tris pH8.0 Make volume up to 200mL with ddH2O
Make volume up to 200mL with ddH2O

Solution 3: Miniprep 0.5M EDTA pH8.0:

120mL 5M Potassium Acetate
23mL Glacial acetic acid
57mL ddH2O
37g EDTA
approx. 3g NaOH
pH to 8.0 with NaOH, The EDTA will
not dissolve until the correct pH is
reached, this may take a while.
Make volume up to 200mL with ddH2O

TE: PEG Solution:

5mL 1M Tris pH8.0 13g PEG 8000
1mL 0.5M EDTA pH8.0 1mL 1M MgCl2
Make volume up to 500mL with ddH2O Make volume up to 50mL with ddH2O

SEVAG: 1M Tris pH8.0:

1mL Isoamyl alcohol 24.22g Tris base in 160mL ddH2O
24mL Chloroform Adjust to pH 8.0 with conc. HCl
Make volume up to 200mL with ddH2O
 90

0.2M HCl: ExoIII Buffer:

17.2mL 36% HCl in 1L 1M MgCl2 3.3µL
20mL 32% HCl in 1L (32% ~ 10M) 1M Tris (pH8.0) 75µL
Make volume up to 1L Make volume up to 5mL

Denaturation solution: Neutralisation solution:

1.5M NaCl 87.66g 1.5M NaCl 87.66g
0.5M NaOH 20g 0.5M Tris HCl 60.55g
Make volume up to 1L pH to 7.2 with HCl, Make volume up to 1L

Mung bean nuclease 50x TAE:

buffer: 96.6g TRIS
1M NaCl 2mL 40mL EDTA (0.5M)
3M Na Acetate 125µL 28.8mL Acetic Acid
1m ZnSO4 125µL pH to 7.6 with acetic acid make
Glycerol 625µL volume to 500mL
ddH2O 2125µL

10x TBE: STE:

108g TRIS 1mL NaCL (1M)
55g Boric Acid 100µL Tris (pH8.0, 1M)
40mL EDTA (0.5M) 20µL EDTA (0.5M)
pH 8.2-8.4, Make volume to 1L Make volume up to 10mL with ddH2O

20x SSC: Fill in Buffer:

175.3g NaCl 0.5M Tris (pH7.5), 0.1M MgCl2, 10mM DTT,
88.2g Na Citrate 0.5mg/mL BSA, 200µM dNTP
pH to 7.0 with HCl, make volume to 1L 1mL Tris (1M, ph7.5)
200µL MgCl2 (1M)
RNase: 20µL DTT (1M)
Disolve RNase to 10mg/mL 100µL BSA (10mg/mL)
in 10mM Tris (pH7.5) 160µL dNTP (2500µM)
with 15mM NaCl 520µL ddH2O
Boil for 10min

ATP Stock: Sephadex G-50:

Dissolve 1g in 10mL ddH2O
pH to 7.0 with NaOH (0.1M)
Volume to 16.67mL and filter sterilise
Add powder to ddH2O (approx. 10g
to 200mL)
Allow to swell, wash 2 to 3 times in ddH2O
Wash 2 to 3 times in TE (pH8.0), store at
4°C
 91

Phosphate Buffer 0.2M: Ferricyanide 0.1M:

A. NaH2PO4.H2O (0.2M) 31.2g/L 4.224g/100mL
B. Na2HPO4 (0.2M) 28.39g/L Ferrocyanide 0.1M:
Add 39mL of A to 61mL of B 3.292g/100mL

GUS assay buffer: 1M Sodium Phosphate

50mL 0.2M phosphate buffer
500µL 0.1M Ferricyanide
500µL 0.1M Ferrocyanide
2mL 0.5M EDTA
60µL Triton X-100
100mg X-gluc (dissolved in ~1mL DMSO)
Make volume to 100mL with ddH2O
Buffer pH 7.2:
68.4mL 1M Na2HPO4 (141.95g/L)
31.6mL 1M NaH2PO4 (156g/L)
Church Buffer:
10mL 1M Sodium Phosphate Buffer
10mL 10% (w/v) SDS

Cracking buffer: Phenol:

1M Tris-HCl, pH 7.5 10mL Wear Gloves throught out procedure
500 mM EDTA, pH 8.0 20mL 1. Melt 500g of phenol at 68°C for 30min
Glycerol 5mL Perform the remaing operations in the
5M NaCl 2mL fume hood and wear gloves
10% SDS 1mL 2. Add 0.5g Hydroxyquinoline
Bromophenol Blue 0.004g 3. Transfer phenol into a 1L Schott bottle
MilliQ H20 62mL 4. Place in stirrer bar
100mL 5. Place bottle on magnetic stirrer
6. Add 500mL 0.5M Tris pH8.0
7. Stir for 15min
8. When phases have seperated, aspirate
the top layer
9. Add 500mL 0.1M Tris pH8.0
10. Stir for 15min
11. When phases have seperated, aspirate
the top layer
12. Repeat steps 9 to 11 3 times
13. Add 50-100mL 0.1M Tris pH8.0
14. Store at -20°C
 92

MS Media
MS Vitamins 1000X
100mL 250mL
Thiamine HCl 10mg 25mg
Pyridoxine HCl 50mg 125mg
Nicotinic acid 50mg 125mg Heat gently and stir to dissolve
Glycine 200mg 500mg
Myo-inositol 10g 25g
water 95mL 237.5mL

MSO pH to 6.0 with 1M KOH, dilute KOH for small amounts.

(Don’t worry, calculations have been checked)
100mL 250 500 750 1000 1250 1500 1750 2000
MS 0.44g 1.09 2.17 3.25 4.33 5.42 6.5 7.58 8.66
Salts

MS Vits 100µL 250 500 750 1000 1250 1500 1750 2000

Sugar 2g 5 10 15 20 25 30 35 40
Agar 0.8g 2 4 6 8 10 12 14 16
0.8%

For rooting use ½ salts and vits, full strength sugar:

100mL 250 500 750 1000 1250 1500 1750 2000
MS 0.22g 0.55 1.09 1.63 2.17 2.71 3.25 3.79 4.33
Salts

MS Vits 50µL 125 250 375 500 625 750 875 1000

Sugar 2g 5 10 15 20 25 30 35 40
Agar 0.6g 1.5 3 4.5 6 7.5 9 10.5 12
0.6%
 93

Appendix C: Antibiotic Solutions Chart:

Antibiotic Working Stock Solubility Storage

Conc. Conc.
(µg/mL) (mg/mL)
Ampicillin 50 Liquid 50 Water -20°C
100 Plates
Carbenicillin 300 50 Water -20°C

Cefotaxime 300-500 250 Water -20°C

Chloramphenicol 170 25 Ethanol -20°C

Gentamycin 25 50 Water -20°C

Kanamycin 50 100 Water -20°C

Rifampicin 50 50 DMSO -20°C

Streptomycin 25 25 Water -20°C

Timintin 500 150 Water -20°C

Geneticin 50 50 Ready 4°C

Made
Tetracycline 10 E. coli 5 50% -20°C
2 Agro Ethanol
 94

Appendix D: Looking after Your Gilson

5 Basic Maintenance Tips:
1. Wind pipette down as far as it will go, pipette up some water and weigh it to perform a
gross calibration. (x4)
2. Then do the reverse by winding it to the max. (x4)
3. Make sure that the shaft is not scratched.
4. At the Maximum volume draw up some water and then check to see if it leaks by
holding it upright.
5. Check that the piston is clean

Cleaning:
• Clean with any cleaning solution then followed by a rinse in a solution of 20-30%
isopropanol is recommended

Operation:
• Add pipette on, Not by jamming it up and down onto it, but by using a slight twisting
motion to ensure a positive airtight seal
• Release the push button SLOWLY and SMOOTHLY to aspirate/dispense the sample
• Wait 1 second and then withdraw the tip from the liquid.
• When dispensing press the push button slowly to the first stop, wait a second and the
to second stop to expel any remaining liquid
• Always release the push button slowly
• ADJUSTING VOLUMES
• increasing volume go 1/3 turn past up and then back to required volume.
• decrease volume go 1/3 past down then back to required volume.
• Leave pipettes hanging vertically after use

Troubleshooting
Check the following
• The connecting nut is loose:
Tighten the connecting nut
• The shaft is cracked or scored:
Remove the tip ejector and inspect the shaft, if damaged replace
 95

• For chemical damage to the piston or seals:

Replace and wash with distilled water
• Improper assembly
Reassemble

If there is liquid in the shaft

Clean the pipette as follows:
• Remove the tip ejector. Unscrew the connecting nut and rinse the shaft, piston, seal and
O-ring with distilled water. Dry these parts and re-assemble the pipette.
• Note the shaft can be autoclaved is necessary, but not the seal and O-ring

If there is an air bubble when sample is aspirated:

• Eject the sample into its original vessel. Check the tip is properly immersed in the sample
liquid
• Pipette the sample ore slowly
If the bubble appears for a second time, replace the tip

Never oil or grease a Gilson

If the tip ejector is loose;

Take out with a pair of pliers, squeeze gap between the 2 metal prongs closer.
 96

CALIBRATION
• Only calibrate if you have checked everything first
• The tolerances for the Gilson pipettes are in the table below
• Calibrate the Gilson at 20% of normal volume. Use distilled deionised water. Then check
at full volume.
• To calibrate, it is best to have someone show you the procedure.
• You do not need to recalibrate a Gilson after dismantling it to clean it.
• You never need to calibrate your Gilson unless it warrants it; ie. 5 years could go past
before it becomes inaccurate.

Gilson Volume Acceptable

µL Accuracy +/- µL
P10 1 0.025
5 0.075
10 0.1
P20 2 0.1
5 0.1
20 0.1
P200 50 0.5
100 0.8
200 1.6
P1000 200 3.0
500 4.0
1000 8.0
P5000 1000 12
2000 12
5000 30