Forward – 5’GAC GGA TCC ACG CAT TAA AGT ATA TTG TGT 3’ (Tm- 66.2 °C) 30bp (with Bamh1 site)
Reverse - 5’ GCC TCT AGA AAT TAA TGA GCG CTT TAA ACT 3’ (Tm- 65.2 °C) 30 bp (with Xba1 site)
My gene size is around 1,300bp.
I tried using a lower temperature (45-50 °C) since the actual annealing portion of the
Fwd primer: ACG CAT TAA AGT ATA TTG TGT, 21 bp long, 28.6 % GC, Tm 55.4 °C
Rev primer: AAT TAA TGA GCG CTT TAA ACT, 21 bp long, 28.6 % GC, Tm 56.2 °C
but got no amplification .
Designing new primers is one of the options but before that I want to try using the old ones. Any suggestions on what changes can be made in the PCR conditions to get the desired results?