3.2 Example

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 :

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:

  1. The mixture was heated at 96C 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;
  2. Denaturation, heated at 96C for 30 seconds. For each cycle, this is usually enough time for the DNA to denature.
  3. Annealing at 68C for 30 seconds: as the temperature decreases the primers are able to anneal to the DNA;
  4. Elongation by heating 72C for 45 seconds: this is the optimum working temperature for the polymerase;
  5. Steps 2-4 were repeated 25 times;
  6. The reaction was held at 7C. 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 7C after just one night.
DNA gel photograph showing the PCR products produced by primers designed from the insulin-like growth factor gene.
Figure 6: PCR of insulin-like growth factor

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.

Cells_selfassessment Design your own PCR program

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