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?

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