Question

After a PCR run using a new set of primers, upon electrophoresis, your gel shows no amplification bands at all. Suggest three changes to the primers you could make and explain why these changes might lead to a better result.

Answer 1

In order to optimize primers the following things can be taken into account like

1.The GC content of the primer should be checked and should be made around 40 to 50% as it affects primer annealing.Also as GC pairing is stronger than AT so the last base should be either G or C to provide stability to primer.

2.The primer sequence should be checked and there should not be any self complementary in the primer to prevent its folding on itself or the reverse and forward primer should not have any complementary to prevent primer dimer formations.

3.The annealing temperature of the primer should be optimum and should be 5 degrees below the melting temperature.