2-Day training on DNA isolation and PCR (7-8 August, 2015)

Genomic DNA isolation

 About 200 mg of plant tissue was ground to a fine paste in approximately 500 μl
of CTAB buffer.
 Transferred CTAB/plant extract mixture to a microfuge tube and incubated the
CTAB/plant extract mixture for about 15 min at 55o C in a recirculating water
bath.
 After incubation, spin the CTAB/plant extract mixture at 12000 g for 5 min to
spin down cell debris.
 Transferred the supernatant to clean microfuge tubes. To each tube 250 μl of
Chloroform : Iso Amyl Alcohol (24:1) was added and mixed the solution by
inversion. After mixing, spin the tubes at 13000 rpm for 1 min.
 Transferred the upper aqueous phase (contains the DNA) to a clean microfuge
tube.
 To each tube 50 μl of 7.5 M Ammonium Acetate was added followed by 500 μl of
ice cold absolute ethanol. Inverted the tubes slowly several times to precipitate the
DNA.
 Tubes were placed for 1 hr at -20 o C after the addition of ethanol to precipitate
the DNA. Precipitates were isolated by spinning the tube at 13000 rpm for 10
minutes to form a pellet.
 Removed the supernatant and washed the DNA pellet by adding two changes of
ice cold 70 % ethanol.
 After the wash, spin the DNA into a pellet by centrifuging at 13000 rpm for 1
min. Removed all the supernatant and allowed the DNA pellet to dry
(approximately 15 min).
 Resuspend the DNA in sterile DNase free water (approximately 50-400 μl H2O).
After resuspension, the DNA was incubated at 65 oC for 20 min to destroy any
DNases and stored at 4 o C.
 Purified DNA was stored at -40 °C and absorbance was noted on Gel Quant pro
(Amersham Biosciences). Quantity of the DNA was determined by measuring
absorbance at 260 nm and 280 nm (A260/A280). Integrity of DNA was checked on
0.8 % agarose gel. The gel was stained in ethidium bromide staining solution and
documented on Syngene gel documentation system (Syngene, UK).

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 2-Day training on DNA isolation and PCR (7-8 August, 2015)

Solution preparation:
CTAB: for 1L of CTAB buffer
100 ml of 1 M Tris, pH 8.0
280 ml of 5 M NaCl
40 ml of 0.5 M EDTA
20 g of CTAB (Cetyl Trimethyl Ammonium Bromide)
to 1L with H20
1 M Tris, pH 8.0: for 1 L
121.1 g Tris
700 ml ddH2O
Dissolve tris and bring to 900 ml.
0.5 M EDTA pH 8.0: for 1 L
186.12 g of EDTA
750 ml ddH2O
Add about 20 g of NaOH pellets
Slowly add more NaOH until pH is 8.0, EDTA will not dissolve
until the pH is near 8.0. 5 M NaCl: for 1 L
292.2 g of NaCl
700 ml ddH2O
Dissolve and bring to 1 L.
7.5 M Ammonium
acetate: for 250 ml 144.5 g ammonium
acetate
Bring to volume with ddH20
Chloroform : Iso Amyl Alcohol (24:1)

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 2-Day training on DNA isolation and PCR (7-8 August, 2015)

Polymerase Chain Reaction (PCR)

PCR
PCR is the cornerstone of a range of modern scientific disciplines and it is also a key
procedure in numerous basic studies involving DNA molecules. All methods for PCR are
based on the concept of DNA strand complementarity discovered by James Watson and
Francis Crick. PCR became possible with the discovery of DNA polymerases and later
their thermostable variants, a variety of isothermal and temperature-cycling amplification
techniques have been developed. Little research related to DNA can be performed
without the employment of PCR or other DNA amplification procedures.

Amplification process is performed in a thermocycler.

PCR amplification reaction mixture:
The amplification reactions with appropriate primers are carried out in a final volume of
50 μl of reaction mixture which includes following composition of reagents:

Serial Quantity for 50 µL Final

Reagents
No. of reaction mixture concentration
1 10 X PCR Taq buffer 5 μL 1X
2 2 mM dNTPs 5 μL 0.2 mM of each
3 Primer I (10 μM) 1 μL 0.2 µM (0.05–1 µM)
4 Primer II (10 μM) 1 μL 0.2 µM (0.05–1 µM)
5 Taq DNA polymerase (u/μL) 0.3-0.5 μL 1.25 u/50 µL
6 25 mM MgCl2 Variable 1 – 4 mM
7 Template DNA Variable 1 μg
To make volume 50
8 Sterile deionized H2O Variable
µL

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 2-Day training on DNA isolation and PCR (7-8 August, 2015)

The PCR cycling conditions:

a. Initial Denaturation Step

The complete denaturation of the DNA template at the start of the PCR reaction is of
key importance. Incomplete denaturation of DNA results in the inefficient utilization
of template in the first amplification cycle and in a poor yield of PCR product. The
initial denaturation should be performed over an interval of 3-5 min at 95 ºC. It can be
extended up to 10 min. Initial heating of the PCR mixture for 5 minutes at 94–95°C is
enough to completely denature complex genomic DNA so that the primers can anneal
to the template as the reaction mix is cooled. If the template DNA is only partially
denatured, it will tend to “snap-back” very quickly, preventing efficient primer
annealing and extension, or leading to “self-priming,” which can lead to false-positive
results.

b. Denaturation Step

Usually 30 sec to 2 min denaturation at 94-95 ºC is sufficient, but this must be

adapted for the thermal cycler and tubes being used (For example, longer times are
required for denaturation in 500 µl tubes than in 200 µl tubes). If the denaturation
temperature is too low, the incompletely melted DNA “snaps-back” as described
earlier, thus giving no access to the primers. Since the PCR product synthesized in the
first amplification cycle is significantly shorter than the template DNA and is
completely denatured under these conditions. If the amplified DNA has a very high
GC content, denaturation time may be increased up to 3-4 min.3

Note: Never use a longer denaturation time than absolutely required for complete
denaturation of template DNA. Unnecessarily long denaturation times decrease the
activity of Taq DNA Polymerase.

c. Primer Annealing Step

For most purposes, annealing temperature has to be optimized empirically. The

choice of the primer annealing temperature is probably the most critical factor in
designing a high specificity PCR. If the temperature is too high, no annealing occurs,
but if it is too low, non-specific annealing will increase dramatically. Primer-dimers
will form if the primers have one or more complementary bases so that base pairing
between the 3' ends of the two primers can occur. Usually the optimal annealing
temperature is 5 ºC lower than the melting temperature (Tm) of primer-template DNA
duplex. Incubation for 30 sec to 2 min is usually sufficient. However, if nonspecific
PCR products are obtained in addition to the expected product, the annealing
temperature should be optimized by increasing it stepwise by 1-2 ºC.

d. Extension Step

Usually the extension step is performed at 72 ºC. The rate to DNA synthesis by Taq
DNA Polymerase is highest at this temperature (2-4 kb/min), and a 1 min extension
time is sufficient for the synthesis of PCR fragments up to 2 kb. When larger DNA

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 2-Day training on DNA isolation and PCR (7-8 August, 2015)

fragments are amplified, the extension time is usually increased by 1 min for each
1000 bp. For fragments up to 3 kb, primer extension is normally carried out at 72 °C.
Taq DNA Polymerase can add approximately 60 bases per second at 72 °C. A 45-
second extension is sufficient for fragments up to 1 kb. For extension of fragments up
to 3 kb, allow about 45 seconds per kb. Cycle extension allows the enzyme more time
to do its job, because as PCR progresses, there is more template to amplify and less
enzyme (due to denaturation during the prolonged high PCR temperatures) to do the
extension.

e. Number of Cycles

The number of PCR cycles depends on the amount of template DNA in the reaction
mix and on the expected yield of the PCR product. For less than 10 copies of template
DNA, 40 cycles should be performed. If the initial quantity of template DNA is
higher, 25-35 cycles are usually sufficient. In an optimal reaction, less than 10
template molecules can be amplified in less than 40 cycles to a product that is easily
detectable on a gel stained with ethidium bromide. Most PCRs should therefore,
include only 25 to 35 cycles. As cycle number increases, nonspecific products can
accumulate.

f. Final Extension Step

After the last cycle, the samples are usually incubated at 72 ºC for 5-15 min to fill-in
the protruding ends of newly synthesized PCR products. Also during this step, the
terminal transferase activity of Taq DNA Polymerase adds extra ‘A’ nucleotides to
the 3/-ends of PCR products. Therefore, if PCR fragments are to be cloned into T/A
cloning vectors, this step can be extended.

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 2-Day training on DNA isolation and PCR (7-8 August, 2015)

Agarose Gel Electrophoresis (AGE)

Electrophoresis is a method of separating charged molecules (DNA, RNA and protein)
based on the rate of movement under the influence of an electric field.
Principle
Electrophoresis working is based on the movement of charged
molecules placed in an electric field toward either the positive
or negative pole according to their charge. During
electrophoresis, the gel is submersed in a chamber containing a
buffer solution and a positive and negative electrode. The DNA
to be analyzed is pulled through the pores of the gel by the
electrical current. Under an electrical field, DNA will move to
the positive electrode (red) and away from the negative
electrode (black). Several factors influence how fast the DNA
moves, including;
 The strength of the electrical field
 The concentration of agarose
 The size of the DNA molecules. Smaller DNA molecules
move through the agarose faster than larger molecules.
Components
The equipment and supplies necessary for conducting agarose gel electrophoresis are
relatively simple and include:
 An electrophoresis chamber and power supply
 Gel casting trays
 Sample combs
 Electrophoresis buffer
 Ethidium bromide
 Loading dye
 Trans-illuminator
Electrophoresis Buffer
Several different buffers have been recommended for electrophoresis of DNA. The most
commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-
EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due
to differences in ionic strength. Buffers not only establish a pH, but provide ions to
support conductivity. If you mistakenly use water instead of buffer, there will be
essentially no migration of DNA in the gel. Conversely, if you use concentrated buffer
(e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.

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 2-Day training on DNA isolation and PCR (7-8 August, 2015)

Ethidium Bromide
Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids
and allows very easy detection of DNA fragments in gels. It incorporates into agarose
gels to enable visualization of the fragments within the gel. Most commonly, ethidium
bromide is added to the gel (final concentration 0.5 µg/mL) at this point to facilitate
visualization.
Agarose
Agarose is a polysaccharide purified from sea-weed. It is also
non-toxic. Agarose gels have a large range of separation, but
relatively low resolving power. It is typically used at
concentrations of 0.5 to 2 %. The higher the Agarose
concentration the "stiffer" the gel will be. By varying the
concentration of agarose, fragments of DNA from about 200 to
50,000 bp can be separated using standard electrophoretic
techniques. An agarose gel is created by suspending dry agarose
in a buffer solution, boiling until the solution becomes clear and
then pouring it into a casting tray and allowing it to cool. The
result is a flexible gelatin-like slab.
Loading/ tracking dye
Loading dye is mixed with DNA samples for use in agarose gel
electrophoresis. It generally contains a dye to assess how "fast"
gel is running and a reagent to make samples denser than the
running buffer. Commonly used dyes are bromophenol blue and
xylene cyanol.
Standards
DNA and protein standards are used in electrophoresis to guide
about the length and molecular weight of the respective sample.
These standards are the known-sequences of bands that can be
compared to unknown samples.
Transilluminator
It is an ultraviolet lightbox which is used to visualize ethidium bromide-stained DNA in
gels.
NOTE: always wear protective eyewear when observing DNA on a trans-illuminator to
prevent damage to the eyes from UV light.
Protocol for 1% agarose gel
 Weigh out 0.5g of agarose into a 250 mL conical flask, add 50 mL of 0.5X TBE
buffer and swirl to mix.
 Microwave for about 1 minute to dissolve the agarose.
 Leave it to cool on the bench for 5 minutes down to about 60 °C (just too hot to keep
holding in bare hands).

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 2-Day training on DNA isolation and PCR (7-8 August, 2015)

 Add 1µL of ethidium bromide (10 mg/mL) and swirl to mix.

 Pour the gel slowly into the tank. Push any bubbles away to the side using a
disposable tip. Insert the comb and double check that it is correctly positioned.
 Leave to set for at least 30 minutes, preferably 1 hour, with the lid on if possible.
 Pour 0.5X TBE buffer into the gel tank to submerge the gel to 2-5mm depth. This is
the running buffer.
Preparing the samples
 Transfer 5 µL of each sample to a fresh microfuge tube.
 Ad 1µL loading buffer into each tube and leave the tip in the tube.
 Load the first well with marker.
 Continue loading the samples and finish of with a final lane of marker.
 Run the gel at 5 V/cm.
 Check that a current is flowing.
 Monitor the progress of the gel by reference to the marker dye.
 Switch off and unplug the gel tank and carry the gel (in its holder if possible) to the
dark-room to look at on the UV light-box.
Composition of 50x TAE buffer
50x TAE buffer is composed of 2 M Tris-Acetate, 0.05 M EDTA, pH 8.3.
Trouble-shooting of Agarose gel electrophoresis
Problem Reason Trouble-shooting
If you see faint or no bands on the  insufficient quantity or  Increase the amount of DNA
gel concentration of DNA but don't exceed 50 ng/band.
 DNA was degraded  Avoid nuclease contamination
 DNA was electrophoresed off  use a lower voltage, or use a
the gel higher percent gel.
 Improper UV light source  Use a
shortwavelength (254 nm) W
light for greater sensitivity
If you see smeared DNA bands  DNA was degraded  Avoid nuclease contamination
 Too much DNA was loaded  Decrease the amount of DNA.
 Improper electrophoresis  Use optimized conditions
conditions  Use ethanol precipitation to
 too much salt in the DNA remove excess salts, prior to
 Small DNA bands diffused electrophoresis.
during satining  Add the ethidium bromide
during electrophoresis.
If you see anomalies DNA band  Improper electrophoresis  Use optimized conditio
migration conditions  Dilute DNA standards in buffer
 DNA was denatured with 20 mM NaCl. Do not heat
standards

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