Question

After designing PCR primers, what next steps should be done? Explain briefly.

Answer 1

Designing the PCR is one of the initial step of PCR

Once the forward and reverse primers are ready we can move to the main steps of PCR reaction which is as follows.

1. Load the DNA sample in PCR tubes in required concentration and volume. 100ng/μl is the usual concentration preferred.

2. In another tube with required concentration add PCR buffer, primers, enzyme and distilled water. This would be the master mix. The volume of each of the components added should be multiplied with the total number of samples so that, it would easier to aliquot the master mix in each PCR tube.

Example if there are 30 samples and the volume of each components required in one tube should be multiplied with 30.

3. Add the required amount of mastermix into each tube.

Example if the total volume is 20 μl and 1 μl is the template DNA sample then add 19μl of master mix into each tube such that each tube will have 20μl in total.

4. Close the cap of the PCR tubes and spin down the master mix to the bottom of the tubes.

5. Programme appropriate thermal protocol in PCR machine and run the reaction. The thermal protocol depends on your experiment.

Example:

Initial Denaturation - 95°C - 2 minutes

Denaturation -  94°C - 30 seconds

Annealing - 60°C - 30 seconds

Extension - 72°C - 30 seconds

Final Extension - 72 °C - 10 minutes

Hold - 10°C

6. After amplification completion, you can resolve the PCR product in 2% agarose gel electrophoresis to check
the amplification.

Thank you!