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Optimization of conventional PCR reactions
A. DNA template:
· Try to use high quality, purified DNA as a template
· When you need to increase fidelity, you can use higher DNA template concentration and reduce the number of cycles
· Template dosage: Take 50 μl reaction system as an example -
Human genomic DNA: 0.1~1.0 μg
E. coli genomic DNA: 10~100 ng
Lambda DNA: 0.5~5 ng
Plasmid or viral DNA: 0.1~10 ng
B. Primer design principles:
· Primer length should meet the specific needs, generally between 18 and 25 bases; when extending long fragments, it is preferably between 24 and 30 bases;
· When introducing a cloning site, an additional 3 or more bases should be added to the end of the primer;
· (G+C)% content should be controlled as much as possible at 40~60%, and the (G+C)% content of the two primers should be as close as possible;
· The GC base distribution in the primer is uniform;
· Try to avoid the same base appearing more than three times in a row, and avoid using A or T at the 3' end;
· Avoid the internal pairing of the primers to form a secondary structure;
· Avoid paired bases between the forward and reverse primers, especially the three bases at the 3' end, otherwise it is easy to generate primer dimers;
· The melting temperature (Tm) of the two primers should be between 42 and 65 ° C, and the best difference between the two primers should not exceed 5 ° C;
· Calculation method of primer Tm value:
Below 20 nt: Tm = 2°C x (A + T) + 4°C x (G + C)
20 nt or more: Tm = 81.5 + 0.41 x (GC%) -600/nt (nt: number of bases of the primer)
· Primer dosage:
· 0.1~1.0 μM, usually starting at 0.2 μM, adjusting the dosage according to the system;
· When using degenerate primers and random primers, increase the total amount of primers to compensate for the loss of yield; however, as the amount of primers increases, the specificity will decrease;
· When the template is larger or more complex (such as human genomic DNA), the amount of primers needs to be reduced to increase the specificity;
· When the template is smaller (such as a plasmid template), increasing the amount of primer can increase the yield.
C. Nucleotides (dNTPs):
· The concentration of conventional dNTPs is 0.1~1.0 mM per nucleotide, usually starting at 0.2 mM, and the dosage is adjusted according to the system;
· Low concentrations (0.05 to 0.1 mM) increase fidelity but reduce yield;
· High concentration increases yield, especially for long-segment PCR, but reduces fidelity.
D. Magnesium ion concentration
· For Taq DNA polymerase, the optimal concentration of magnesium ions is 1.5~2.0 mM;
· The optimum concentration depends on the template, buffer, DNA and dNTPs (each of which may bind magnesium ions);
· The magnesium ion concentration is too low, which will reduce the yield;
· If the magnesium ion concentration is too high, it will increase the non-specific PCR product;
· Optimize magnesium ion concentration, usually in a gradient of 0.5 mM up to 4 mM.
E. Taq DNA polymerase concentration
· It is recommended to use a reaction system with a concentration of 1~2.5 U/50 μl.
F. initial reaction
· Prepare a reaction system on ice;
· Finally add polymerase;
· Preheat the thermocycler to the denaturation temperature (94 ° C), place it in the PCR tube, and immediately react.
G. Denaturation temperature and time
· Initial denaturation at 94 ° C, allowing the DNA duplex to open completely;
· The denaturation time is usually 15~30 seconds;
· Avoid prolonged or high temperature incubation;
· High GC content template can increase the denaturation temperature to 98 °C.
H. Annealing temperature and time
· Normally, the annealing temperature is the primer Tm value minus 5 ° C, between 55 ~ 60 ° C;
· Increasing the annealing temperature helps to reduce non-specific bands;
· The normal annealing time is 15~30 seconds.
I. Extension temperature and time
· The extension reaction is usually carried out at 72 °C.
· The extension time of Taq enzyme is about 15~30 sec/kb DNA;
· When the product is less than 1 kb, the recommended extension time is 30~60 seconds;
· Longer extension times may be required for products greater than 3 kb or for reactions over 30 cycles.
Typical cycling conditions:
Pre-deformation 94 ° C 2 minutes
94 ° C for 30 seconds,
55 ° C for 30 seconds,
72°C 1 minute / 1 kb, 25~30 cycles
72 ° C 5 minutes
Note: The above reaction conditions apply to PCR reactions catalyzed by common Taq DNA polymerase. When the DNA template is rich in GC, has a complex secondary structure, a low concentration, or the product is >5 kb, it may be necessary to change the corresponding conditions.
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