The times and temperatures given in this example were taken from a PCR program that was successfully used on a 250bp fragment of the C-terminus of the insulin-like growth factor gene (IGF).
The reaction mixture consisted of :
- 1.0 μl DNA template (100 ng/μl).
- 2.5 μl of primer, 1.25 μl per primer (100 ng/μl).
- 1.0 μl DNA polymerase.
- 1.0 μl nucleotides.
- 5.0 μl buffer solution.
- 89.5 μl water.
A 200 μl reaction tube containing the 100 μl mixture was placed in the PCR machine. Four reactions were set up, tubes 1-3 contained DNA from different tissues and tube 4 contained DNA from the gene itself as a positive control.
For this reaction the PCR process consisted of the following steps:
- The mixture was heated at 96°C for 5 minutes to ensure that the DNA strands as well as the primers have been denatured. The DNA polymerase was present at this stage, it could have been added after this step;
- Denaturation, heated at 96°C for 30 seconds. For each cycle, this is usually enough time for the DNA to denature.
- Annealing at 68°C for 30 seconds: as the temperature decreases the primers are able to anneal to the DNA;
- Elongation by heating 72°C for 45 seconds: this is the optimum working temperature for the polymerase;
- Steps 2-4 were repeated 25 times;
- The reaction was held at 7°C. This is useful if you start the PCR in the evening just before leaving the lab, so it can run overnight. The DNA will not be damaged at 7°C after just one night.
In Figure 6 a set of specific primers designed to produce a 250 bp fragment from the insulin-like growth factor gene were used on a set of three different tissues. Tissue 1 does not have the gene but Tissue 2 and Tissue 3 do as can be seen by the presence of a fragment corresponding to that in the positive control.