i hv a small doubt with my pcr primers. i hv to amplify a fragment of 54 bp, for which i designed a set of 10 nucleotide primers. but the melting temperature of the primers is 30 degrees C. i hv a doubt if such a low melting temperature will interfere in the amplification. i also want to know if anyone has amplified a small fragment like tis. pls help with the preferrable extension time for the primers. kindly help…..
The problem with using such low binding temperature primers is two fold. the first is Taq does not have much activity at 30C. The second is the template will have a fair amount of secondary structure that prevents the primer binding. The experiments we have done developing UniSeq showed that you really want to stay above 37C annealing temp and also have at least 11 bases binding.
thank you for the reply. i was advised to get my 54 bases as chemically synthesised oligonucleotides. but am not sure how this works. can u pls make tis clear?
If you want to make a 54 base oligo just contact a company that makes them and give them the sequence you want. They will make it and ship it to you.
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