I am conducting a project which involves introducing the L812M Mutation into GRIN2A through site directed mutagenesis.

I have been struggling with the mutagensis PCR, as only very faint bands are produced when the pcr product is ran on a gel.

I suspect my PCR conditions are not optimised properly and would like help optimising the pcr as i suspect that to be the problem (particularly the annealing temperature and extension). The Tm of my primers are 69, 35-ber and the plasmid I am using is 15kb in length.

The previous PCR conditions that resulted in very weak bands were,

Segment 1 (1 cycle) Initial Denaturation/95°C/30 sec

Segment 2 (16-18 cycles) Denaturation/95°C/30 sec

Annealing/65°C 1 min

Extension/68°C 15 min (1 min/kb for 15kb plasmid as instructed in protocol)

Segment 3 (1 cycle)Final Extension 16 8°C 10 min

Segment 4 Final Hold 14°C ∞

I would like to note that I was following a protocol to do this site directed mutagenesis from agilent which instructed to do an extension at that given temperature at 1min/kb of plasmid.

Any help would be greatly appreciated :)