PCR

Polymerase Chain Reaction

1.Add the following to a microfuge tube:

10 ul reaction buffer

1 ul 15 uM forward primer

1 ul 15 uM reverse primer

1 ul template DNA

5 ul 2 mM dNTP

8 ul 25 mM MgCl2or MgSO4(volume variable)

water (to make up to 100 ul)

2.Place tube in a thermocycler.

Heat sample to 95°C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.

3.Start the PCR cycles according the following schemes:

(1) denaturation - 94 ° C, 30-90 sec.

(2) annealing - 55 °C (or -5° Tm), 0.5-2 min.

(3) extension - 72 °C, 1 min. (time depends on length of PCR product and enzyme used)

(4)repeat cycles 29 times

4.Add a final extension step of 5 min.

to fill in any uncompleted polymerisation. Then cooled down to 4- 25 °C.

Note:

Most of the parameters can be varied to optimise the PCR:

(a)Mg2+ one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.

(b)Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.

(c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity . Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloningbut needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work.