Hello, I am using expression primers having restriction sites plus extra 4 base pairs, I have tried gradient PCR with Dream Taq as well as Q5 DNA polymerase but there has been no amplification so far.. please tell me how DMSO would help with optimizing annealing temp. ? at which range of temp should I use? Tm according to neb.calculator is 57 and 56, at which I have tried already.. but nothing seems to work
Hi
Set the gradient PCR with annealing temperature from 50-60 degrees.... DMSO generally used for amplifying GC rich amolicons.
Hi there,
If you are sure about the design of the primer set and the PCR protocol then the problem is the DNA template. Indeed addition of DMSO should improve the annealing of primers to the target DNA. What I usually do is to use exactly the same PCR protocol with a sample containing 5%DMSO.
Hi
I would do a Touch down PCR from 60 to 50° (10 cycles ) and 20 to 30 more cycles at 50°C. DMSO can help if the region to amplify is GC rich.
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