Title: Plasmid Extraction, DNA Quantitation and Restriction Digestion

Aim:
Pm e

Kjkg/llhli

Dna q
Hjvjjm

restr
Method:
Method for Plasmid Extraction:
1. (This will be done for you.) Grow E. coli cells overnight in LB broth with 100 µg/ml
ampicillin (ampicillin is the antibiotic commonly used as it is very stable in agar plates and it’s a
broad-spectrum antibiotic that 1. (This will be done for you.) Grow E. coli cells overnight in LB
broth with 100 µg/ml of ampicillin (ampicillin is the antibiotic commonly used as it is very
stable in agar plates and it’s a broadspectrum antibiotic that kills a wide variety of bacteria which
results in infrequent plate contamination).
2. Transfer to a 1.7 ml microfuge tube – you will collect your microfuge tube - and centrifuge for
2
min. Decant supernatant to remove most of the growth media
3. Repeat step two.
4. Resuspend pellet thoroughly in a 100 µl of TEG (solution 1) and leave it on ice for 5 min.
5. Add 100 µl of solution 2, mix and leave 5 min. at room temperature for cell wall lysis.
6. Add 400 µl of solution 3, shake hard and let mixture stand on ice for 5 min.
7. Add 300 µl of solution 4, mix by inversion twice.
8. Place tube on ice for 5 min., mix by inversion every 2 min.
9. Centrifuge for 5 min.
10. Transfer 750 µl of the clear supernatant to a new 1.7 ml microfuge tube.
11. Precipitate DNA by adding 0.6 volume of isopropanol, mix by inversion and equilibrate at
room
temperature for 10 min.
12. Centrifuge for 5 min. to pellet the DNA.
13. Pour off the isopropanol or use a pipette to remove the isopropanol leaving approximately 50
µl.
14. Add 200 µl of 70% ethanol, then gently flick the tube to wash the pellet.
15. Centrifuge for 3 min., decant supernatant, blot tube on a clean paper towel and allow the
pellet to
air dry on the bench.
16. Resuspend DNA pellet in 20 µl of TE. Pool the extracts for each bay [ensure the tube is
properly
labelled]. The Tris will buffer the DNA while the EDTA will protect the DNA from degradation
by DNases (by binding to the divalent cations which are cofactors for DNase activity).
17. Extracts will be stored at -20°C for the next lab

Methods for DNA Quantitation:

1. (This will be done for you) Prepare serial dilutions of known concentrations of DNA between
50 µg/ml to 0.5 µg/ml.
2. Spot 2 µl of each to on a previously poured 0.8% agarose gel in 0.5 x TBE as indicated by the
instructor to form about a 2 mm circle.
3. Spot 2 µl of your pooled pBR322 DNA extract.
4. Allow the spotted DNA solutions be absorbed by the gel, this should take approximately 15
min. at room temperature.
5. (This will be done for you) Stain the gel in a 1 µg/ml solution of ethidium bromide for 10
min.
6. De-stain in a 1 mM magnesium sulfate solution for 10 min. to remove any background stain.
7. Visualize under UV light and photograph.
8. Compare your extracts to the known standards to estimate the concentration

Methods for Restriction Digestion:

1. Put on a pair of latex gloves. Do not handle tubes or pipettors before – it is possible that your
DNA will contaminate the experiment.
2. Wipe bench area with 70% ethanol provided.
3. Label one 0.75 ml microfuge tube ‘BstN I’ and the other ‘Hind III’ and add the reagents as
indicated below:
4. Mix the contents of each tube by pipetting slowly up and down 2-3 times.
5. Place the tube labelled BstN I to incubate at 60°C and the Hind III tube at 37°C for 1 hr then
store
on ice.
6. Label 3 clean 0.75 ml microfuge tubes pBR322, BstN I and Hind III and add 5 µl loading dye
to
each tube.
7. Place 10 µl of the DNA digest (step 3) in the tubes labelled in step 6. Add 10 µl of undigested
plasmid pBR322 DNA to the remaining tube from step 6.
8. Mix the contents of each tube (DNA and loading dye) by slowly pipetting up and down 2-3
times.
Gel electrophoresis
9. The demonstrators will show you how to load your samples into the agarose gel.
10. The samples will be subjected to electrophoresis at 100V for 30 min in 0.5 x TBE buffer.
11. Carefully slide the gel off the tray and into the staining container.
12. Leave the gel to stain for 5-10 minutes. Destain gel in distilled water for 5 to 20 mins., to
help
remove background ethidium bromide.
13. View and photograph gel under a UV transilluminator
Results:
Electrophoretogram of

1 2 3 4 5 6 7 8 9 10 11 12 13
14141 31313

1kb DNA ladder

kilobases

kilobases

kilobases
8.0 kilobases
kilobases
4.0 kilobases
kilobases
4.0 kilobases
4.0 kilobases
4.0 kilobases
4.0 kilobases

4.0 kilobases 3.5 kilobases

4.0 kilobases
1.9 kilobases

4.0 kilobases

Key
Lane
8 Undigested pBR322
9 BstNI
10 HIndIII
Questions
1. Consider the 3 major classes of biologically important molecules: proteins, lipids and nucleic
acid. Which steps in the miniprep procedure act on proteins (4 marks)? On lipids (3marks)? On
nucleic acids (4 marks)?
SDS-sodium hydroxide solubilizes proteins and lipids, which are precipitated upon the addition
of potassium acetate. Additional proteins are removed into the isopropanol. Both isopropanol and
ethanol precipitate nucleic acid.

2. What other kinds of molecules, in addition to plasmid DNA would you expect to find in the
final miniprep sample (4 marks)?
3. Draw a restriction map of the plasmid pBR322 showing the BstN I and Hind III restriction
sites. (10 marks)

4. What do ApR, TcR, and ori on the pBR322 map represent and discuss their functions? (6
marks)
ApR and TcR represent antibiotic resistance genes (selectable markers)
Allows for selection of plasmid-containing bacteria by providing a survival advantage to the
bacterial host. Each bacterium can contain multiple copies of an individual plasmid and ideally
would replicate these plasmids upon cell division in addition to their own genomic DNA.
Because of this additional replication burden, the rate of bacterial cell division is reduced (i.e., it
takes more time to copy this extra DNA). Because of this reduced fitness, bacteria without
plasmids can replicate faster and out-populate bacteria with plasmids, thus selecting against the
propagation of these plasmids through cell division.
To ensure the retention of plasmid DNA in bacterial populations, an antibiotic resistance gene
(i.e., a gene whose product confers resistance to ampicillin) is included in the plasmid. These
bacteria are then grown in the presence of ampicillin. Under these conditions, there is a selective
pressure to retain the plasmid DNA, despite the added replication burden, as bacteria without the
plasmid DNA would not survive antibiotic treatment. It is important to distinguish that the
antibiotic resistance gene is under the control of a bacterial promoter, and is thus expressed in the
bacteria by the bacterial transcriptional machinery

Ori
This represents the origin of replication
Their origins of replication enable the plasmids to replicate independently of the bacterial cell
cycle. Origins of replication are critical for the ability of the plasmid to be copied (amplified) by
bacteria, which is an important characteristic of why plasmids are convenient and easy to use.

5. Does the undigested plasmid show more than a single band when electrophoresed? Write
short notes explaining why this may occur. (4 marks)
Discussion
Expected & observed band sizes correlate? (5 marks)
Were there any discrepancies noted? (5 marks)
References (4 mark)