primer with higher melting temperature - (May/05/2011 )
i am doing research in cancer. I bought new primers for my work.Tm for tese forwrd and reverse primers are 73.3 and 72.9 respectively. I did PCR with these. but i didnt get any product in any temperature(45-72c).pleare give some suggestions.
if you hav no other option than this primer you can very well try to optimise the condition for this primer itself, but will demand time and tricky....fine what is your template???is it DNA, plasmid or cDNA??? What PCR mix you are using, is it a readymade mastermix or the conventional tag+dntps+mgcl2 etc??? what abt the GC content of your region to be amplified??? if gDNA how pure is your template??? so all these things matters....so answer for all these queries you will find some ways to troubleshoot your pcr in this forum...
Just curious: when you do your gradient from 45-72C, how many temperature difference between each interval? 45C, 46C, 47C, 48C..... or 45C, 50C, 55C....?