I will do a PCR for cloning, for this reason the primers have the sequence of the ERs at their ends, for this reason the Tm are high. It is possible to do a PCR with these high Tm primers and what annelling temperature work?
is your entire primer binding to your target sequence? if yes then you can do a two step PCR in which the extension and annealing step are combined into one step at 72C. If only a portion of your primer is binding then you have to base your annealing temperature based on the binding portion only.
is your entire primer binding to your target sequence? if yes then you can do a two step PCR in which the extension and annealing step are combined into one step at 72C. If only a portion of your primer is binding then you have to base your annealing temperature based on the binding portion only.
Hanna Alalam is correct ...if you have restriction sites and extra bases at the 5' ends of your primers and these bases do not anneal to your genomic sequence then you must not include these bases in your calculation of annealing temperature. After the first few cycles they are incorporated into the pcr product and only then can the annealing temperature be increased
Hot start gold DNA polymerase can amplify at up to 90 degree centigrade temperature. It's possible to do pcr using your primer. I also done a PCR and my primer annealing temperature was 86 degree centigrade.
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