I try to PCR my gene target, that length about 4,000 bp. First time PCR, I try with cDNA 5ng, each concentration primer 0.1mikroMolar, dNTP 0.4mM, KOD FX 1.0U. and I got the smear band, I try to optimize the annealing temperature but I always get the smear band and sometimes I got the smear band in wrong size (below target size). I set my PCR 96C 5min, 96C 30s, 52C/55C/62C 40s, 72C 4min, 4C hold. May anyone help my problem?
Thanks