Me is working on the project in which amplification of SARM ( CDS portion, 2200bp) is needed. I have made the cDNA and designed the primers with additional restriction sites. But I am unable to amplify.
I am using LA taq
Atg gtc ctg acg ctg ctt ctc tcc (31 bp)
Tta ggt tgg acc cat ggg tgc agc (31 bp)
Red colored are the restriction enzyme sites added by me which are not present in my sequence
I have tried below mentioned thermocycling conditions
initial denaturation 95C for 5min
denaturation 95C for 1min
annealing 51 or 53 or 55 or 57 for 1 min
extention 72 for 2 min
and final extenion 72 for 5min
total cylces were 35
Can you please suggest anything ?