So I designed some primers but the melting temperature is all above 70 degree. I know that this should not be done, but to meet all the criteria, there is no other way. My main primer is 21 base (to be sure of specificity), and the ratio of GC is above 50% (its the nature of the gene that I want to amplify). I could have deleted some bases but that would not ensure the specificity.
So is there any better way to lower down the temperature or must I follow and order these primers anyway?
I would make longer primer, allow no more than 5 GC in the last 10 bases in 3'end, at the same time making silence mutant (no change for coding amino acid) change of the GD rich region by change G to A, and C to T. And more important, you need to avoid more than 3 continuous CG in the every 3'end. Such G to A change and C to T change actually count as half match for annealing. They contribute to annealing is as good as A=T matching. In this sense, you may also make a normal length primer, just by this alter silence change.
I would try both. Order the primer with the original temperature and see if it is working. Second I would order some shorter ones (down to 17 bp is also fine). By doing a touch-up PCR (increase temperature) you can work even with a bad specificity in the first cycles. For GC rich sequences add some DMSO to the reaction. This improves a lot!
I would use smaller primers trying to exclude some of GC if it is possible
I would make longer primer, allow no more than 5 GC in the last 10 bases in 3'end, at the same time making silence mutant (no change for coding amino acid) change of the GD rich region by change G to A, and C to T. And more important, you need to avoid more than 3 continuous CG in the every 3'end. Such G to A change and C to T change actually count as half match for annealing. They contribute to annealing is as good as A=T matching. In this sense, you may also make a normal length primer, just by this alter silence change.
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