I did a gradient PCR for 16SrRNA with universal primers 27F and 1492R. The PCR product size is around 400 bp instead of 1.5 kb. What could be the possible reason. the annealing temp. was ranging from 48 to 56 oC. at 30 sec and extension was 72 oC for 1 minute. I prepare 10 ul rxn with 5 ul 2X Red Taq PCR mix, I ul each primer (20 pico mol) and 3 ul DNA isolated through P:C:I method.