Question

What Are Touchdown PCR and Colony PCR?

Answer 1

Touchdown PCR:

1. Touchdown PCR is a variant of PCR.
2. It is a method of polymerase chain reaction by which primers avoid amplifying nonspecific sequences.
3. It increases the specificity of PCR reactions.
4. It involves gradually decreasing one to two degree Celsius per second of the cycle.
5. The initial annealing temperature should be higher than that of the T m (meltdown temperature) of the primers.

Colony PCR:

1. Colony PCR is a convenient method to rapidly screen colonies of transformed bacteria or yeast, grown in a selective medium.
2. This is done to check if the transformed bacterial clones like E.coli contain the desired gene.
3. Selected colonies are taken up using a sterile toothpick from an agarose plate and then swirled into the sterile water.
4. Then the mixture is boiled at 100⁰C for 2 minutes, followed by ultracentrifugation for 2 minutes.
5. Then standard PCR protocol is followed with the supernatant at 95⁰C.