Polymerase Chain Reaction
(PCR)
This protocol will help those who are new to PCR to get started. It will also help any one
who wants to learn more about ways to enhance the basic PCR technique, for enhance to
optimize reactions, prevent carry-over contamination, or extra long amplification product
1) Introduction
Individual nuclear DNA molecule contains hundreds of millions of nucleotides. When
DNA is isolated from cells using standard methods, these huge molecules are fragmented
by sheer forces, generating complex mixtures of still very large DNA fragment.
Given the complexity of the DNA isolated from the cells of a typical eukaryotes, or even
prokaryote, the challenge was how to analyze. So to study the specific DNA sequences
within a complex DNA population, DNA cloning was a fundamental of current DNA
technology. This technology requires selective amplification of specific DNA component
(the target DNA) occurring within a complex starting DNA population, which may be the
total genomic DNA within a particular tissue (or cell type), or complementary DNA
(cDNA) prepared from the total RNA of a particular tissue. Amplification is achieved
using a DNA polymerase and can be carried out in vitro.
The desired target DNA is selected using oligonucleotide primers which will bind
specifically to this sequence. Once the sequence specific primers are bound to the target,
a heat-stable DNA polymerase can generate additional copies of the target sequence. The
new copies of the target in turn serve as templates to make more copies still in chain
reaction which has been named the polymerase chain reaction (PCR).
Getting started in PCR
The primary requirements for a polymerase used in the polymerase chain reaction (PCR)
are good activity at temperatures a round 72°C and ability to retain that activity after
prolonged incubations at even higher temperatures (95°C).
This section describes Taq DNA polymerase which meets these requirements.
Source: Taq DNA polymerase is isolated from the thermophilic eubacterium Thermos
aquaticus a strain which lacks Taq 1 restriction endonuclease.
General properties:

Size and activity: it is a highly processive 5´-3´ DNA polymerase but lucks 3´- 5´
exonuclease activities.

Thermal stability: Taq DNA polymerase has half life of less than 5 minutes at
100°C. However the enzyme retains its activity for much longer time (half life up
to 40 minutes) at 95°C.

Magnesium concentration: Taq DNA polymerase prefers Mgcl2 as metallic
cofactor. The standard magnesium concentration for PCR with the polymerase is
1.5 mM.

dNTPs concentration: the nucleotide concentration during PCR should be 200 μM
for each PCR.

Detergent and other additives:
1- None ionic detergent Tween 20 (0.5-1.0%) has been used to enhance the
efficiency of Taq DNA polymerase in certain PCRs.
2- Other additives reported to have enhancing effects include: DMSO, gelatine,
glycerol and ammonium sulfate.
1-General PCR that works with the common PCR enzyme:
The optimal conditions for PCR (incubation times and temperatures, concentration of
enzyme template DNA, primers and Mg2+ ions) vary. Thus the protocol given below
must be determined for each PCR reaction. It is especially important to titrate the Mg2+
concentration and the amount of enzyme required per assay.
Procedure
1) Briefly centrifuge all reagent s before begging the procedure.
2) setup in a sterile microfuge tube on ice,
Temperature
Time
Number of cycle
Initial denaturation
95ºC
1 min
1 first
Denaturation
95 ºC
30s
Annealing
50-65 ºC
min
Elongation
72 ºC
45-3 min
25-40
Final elongation
72 ºC
7 min
3) Gently vortex the mixture and then centrifuge briefly to collect the sample at the
bottom of the tube.
4) Place your sample in a thermocycler and start PCR.
Modifying the general PCR protocol to improve results:
 The general PCR protocol usually gives satisfactory results. however, many
amplification reaction require titration of magnesium for optimal result. in fact,
the results of changing the Mgcl2 concentration over narrow range can be quite
striking.
 In most cases a concentration of 1.5 m mol/L will produce satisfactory PCR
results with Taq DNA polymerase. In general higher Mg2+ concentration increase
the PCR yield, but degrease the specificity of the reaction (and increase the
incidence of the primer diamers); low Mg2+ concentrations increase the specificity
but decrease the yield. However if the general PCR protocol with Taq DNA
polymerase does not produce an amplification product, use the following protocol
to adjust the Mgcl2 concentration.
The table below gives the volumes of the Mgcl2 stock solution which give the
designated Mgcl2 concentrations when added to a 100µl PCR mixture.
To
reach
a
final
Mg2+ 1
1.25
1.5
1.75
2
2.5
5
5
6
7
8
10
20
concentration (mM) of:
Add this a mount of 25mM Mgcl2 4
solution (µl):
Alternative reaction buffers for PCR
In addition to reagents listed below to devise an alternative reaction buffer. If you
have 4 buffers with different PH this will allow you to optimize both Mg2+ and the PH
of your PCR reaction at any combination of four PCR reaction at any combination of
four different PHs ( from 8.3 to 9.2) and four different Mgcl2 concentrations (from 1.0
to 2.5mM).
Mgcl2 concentration (mM)
PH
1.0
1.5
2.0
2.5
8.3
A
B
C
D
8.6
E
F
G
H
8.9
I
J
K
L
9.2
M
N
O
P
Once you have right combination of PH and Mgcl2, you can use additives to optimize
your reaction further.
 DMSO may help to reduce unspecific priming.
 Gelatin and glycerol stabilize Taq DNA polymerase during PCR to improve
the product yield.
 Ammonium sulfate may also improve the product yield
PCR Master Mix
PCR are as simple as adding primers and template to an aliquot of our PCR master. It
contains
1) Taq DNA polymerase (2.5 units/100 µl reaction).
2) PCR nucleotide mix (0.4mM of each nucleotide
3) Optimized reaction buffer
4) Mgcl2
To minimize pipetting and the potential for contamination, the PCR master mix is
provided in separated vials.
What to do when the PCR does not work?
If a particular PCR protocol does not amplify the desired PCR product, any one of a
thousand things could be responsible.

Consequently, you should check the most obvious trouble points first before
embarking upon a full-scale check of all reagents and solutions

In general if a PCR does not work then something is amiss with the starting
material (genomic DNA, RNA or cDNA), the primers or the thermostable
polymerase.
Step1. Repeat the PCR with the same material (this is a check for pepetting errors or
reagents that were inadvertently omitted in the first PCR.
Step2. Check starting material by making new dilutions of the genomic DNA from stock
solutions stored at -70 ºC or from ethanol precipitates. If fresh working solutions don’t
solve the problem, check the stock solutions by running a sample on an agarose gel (this
analysis reveals wholesale degradation of the DNA.
Step3. Check primer concentrations and primer sequences especially those used for RTPCR, should be checked and re-checked before they are used.
Step4. Check the thermostable polymerase (this is the last resort).
Step5. Start again
To save time, discard all buffer solutions, primer working solutions and sample working
solutions. Re-dilutes primers and samples. Make fresh buffer solutions. If possible order
another bach of thermostable polymerase.
PCR optimization strategies
Although the PCR concept is simple, successful performance of a PCR reaction depends
on a number of factors
Optimizing primer design
The primers should be complementary to particular sites on the template DNA. Each of
the primer should bind to only one of the two template strands. The optimum distance
between primer annealing sites is generally 100-1000bp
General considerations for primer design
1. PCR-primers have a 40-75% G/C-content and contain 18-40 nucleotides.
2. Whenever possible, avoid on imbalanced distribution of G/C and A/T-rich
domains in the primers.
3. If published primer sequences are to be used, determine the primer sequences
from at least two references.
4. Alternatively, suitable primers and primer pairs may be designed manually or
with aid of commercially available computer software.
5. Primers that are used in combination should have a similar Tm value.
6. Usually an equal molar concentration of both primers is required to avoid
asymmetric reaction condition.
2-Restriction Fragment length polymorphisms (RFLP-PCR)
1. Amplify the DNA regions of interest by PCR
2. Add 0.5 µl of the restriction enzyme and 1.5 µl of restriction buffer to 8 µl of
PCR product and mix well.
3. Incubate the amplicons with restriction endonucleases for three hours at 37ºC and
addition 15 min at 65 ºC to inactivate the enzyme. If there is a restriction enzyme
site, the endonuclease cuts the PCR product.
4. The different alleles can be easy differentiating by electrophoresis through
agarose gel, which was stained with ethidium bromide and visualized by UV
light.
3-Reverse transcriptase PCR (RT-PCR)
Synthesis of cDNA
1. . . . RT-reaction with Oligo dT:
Mix 1:
RNA (200mg/ul)- 5ul
1ug
Oligo dT -2ul
50um
Heat at 70ºC for 10min (RT1),chill on ice, spin for sec
Mix 2:
DNTPs
2.5ul
RT-buffer
2.5
RT-enzyme
0.5
RNase inhibitor
0.5
RNase free water
12.5
Mix gently ,then
42ºC-50 min (RT2)
1.
RT-reaction with random hexamers
· 1 mg of RNA (or 0.1 mg of mRNA) in 9.5 ml of H2O
· incubation at 70°C for 10 min
· cool on ice and add other reagents to final volume of 20 ml:
RT buffer: 20 mM Tris HCl, 50 mM KCl, pH 8.3
MgCl2: 5 mM
DTT: 10 mM
random hexamers: 5 mM
RNAasin: 20 units
RT enzyme: 200 units Superscript (200 units per ml)
dNTP: 1 mM
· temperatures and incubation times:
room temperature for 10 min
42°C for 45 min
99°C for 3 min
4°C at end of RT step