Component	Amount (for 100 µl reaction)	Final Concentration
H2O (DNase free)	- µl
10x PCR Buffer	10 µl	1x
dNTP (25 mM of each dNTP)	0.8 µl	0.2 mM of each dNTP
DNA template	- µl	1 pg ~1 µg
Primer 1 (10 µM)	2 µl	0.2 µM
Primer 2 (10 µM)	2 µl	0.2 µM
Taq DNA polymerase (5 U/µl)	0.4 µl	2 Units
PCR Cycling Program	94 oC x 4’ (94 oC x 30”, 55 oC x 30”, 72 oC x 1’),  30 cycles 72 oC x 5’

 

 

Optimization of PCR Compositions

The use of PCR to amplify a single or multiple target sequences (amplicons) often produces unwanted products or no product at all. So the optimization of PCR conditions by changing the composition of one or several of the key variables becomes necessary. Among all the variables that contribute to primer-template fidelity and primer extension, the Mg²⁺ concentration and the annealing temperature are the most important.

 

1. Template DNA

The amount of DNA template required varies according to the type of the DNA to be amplified. Generally, 100 ng to 1000 ng of genomic DNA is recommended for a total PCR reaction volume of 100 µl. Less DNA (0.01-50 ng) can be used for amplification of plasmid DNA, purified DNA, and viral DNA.

2. Magnesium Concentration

The Mg²⁺ concentration varies in different PCR buffer systems. For amplification of genomic DNA sequences, the optimal Mg²⁺ concentration could be determined by adding Mg²⁺ to a series of final concentrations of 1.5 mM to 4.5 mM. Normally lower Mg²⁺ concentration increases the specificity of the amplification products but with lower yield; higher Mg²⁺ concentration increases the yield of non-specific amplification products but promotes misincorporation.

3. Primer Concentration

The primer concentrations between 0.1 µM and 1 µM are generally optimal. Higher primer concentration may promote msipriming and accumulation of nonspecific amplification products.

4. Taq DNA Polymerase Concentration

The amount of Taq DNA polymerase varies according to the length of the templates to be amplified. Successful amplification is usually achieved at 1~2.5 units of Taq/100µl reaction volume (up to 5 units/100µl could be used for long range amplification). Higher Taq DNA polymerase concentrations may cause synthesis of nonspecific products.

Note: Taq DNA polymerases from different suppliers may behave differently due to different formulations, assay conditions, and unit definition.

5. dNTP Concentration

The concentration of each dNTP in the reaction mixture is usually 200 µM. The four dNTPs should be used at the equivalent concentration to minimize the misincorporation errors.

Both the specificity and the fidelity of PCR are increased by using lower dNTP concentration (10-50 µM) than the recommended 200 µM.

6. Primer Annealing

The typical annealing temperature is between 55 ^oC and 72 ^oC. The optimal annealing temperature could be calculated by “Tm - 5 ^oC”.

7. Primer Extension

Usually the extension step is performed at 70~75°C. The rate of DNA synthesis by Taq DNA Polymerase is the highest at this temperature (2~4 kb/min), and a 1 minute extension time is sufficient for the synthesis of PCR fragments up to 2 kb. When larger DNA fragments are amplified, the extension time is usually increased by 1 minute for each additional 1kb. 

8. Hot Start PCR

The Hot-Start activation method has been increasingly used to improve the performance of PCR, especially for multiplex PCR and real-time PCR. The Hot-Start method is to block DNA polymerase extension at the lower temperatures (4 ^oC ~ 25 ^oC) before the initial denaturation temperature in PCR is reached.