PS: Redesigning of primers isn't feasible.
The temperature difference is about 11 °C with GC content 63% in forward and 37% in reverse primer.
pcr depends on the lower annealing primer being able to anneal so run an annealing temperature gradient from lower annealing -2 to +10 degrees and see what works. If you get too many bands in your best pcr then do the same with up to 5% dmso added to the reaction mix. In spite of text book advice I have often found mismatched primers to be very forgiving in generating the correct amplimer and band purification from agarose is always an option. Also if your gene is GC rich then add a final concentration of 1M betaine to help it to melt and amplify better
pcr depends on the lower annealing primer being able to anneal so run an annealing temperature gradient from lower annealing -2 to +10 degrees and see what works. If you get too many bands in your best pcr then do the same with up to 5% dmso added to the reaction mix. In spite of text book advice I have often found mismatched primers to be very forgiving in generating the correct amplimer and band purification from agarose is always an option. Also if your gene is GC rich then add a final concentration of 1M betaine to help it to melt and amplify better
also consider touchdown pcr based on the lower annealing oligo if you get multiple bands
- Badges
- Science method