I am trying to use overlap extension PCR to combine 2 linear PCR fragments around 1kb each. I amplified both fragments with overhanging primers with a 20 bp overlap between the two fragments. When I do overlap extension PCR, I just get amplification of the individual PCR fragments. I am doing a PCR reaction for 15 cycles without the primers, and then adding the primers that flank either end of the combined product for another 15 cycles.
Does anyone have suggestions for troubleshooting? The overlap region between the two fragments has a TM of 54, and the primers have TMs of 74 and 78. For the overlap PCR reaction I tried an annealing temperature of 50 and 55, and for the extension reaction I have tried annealing temperatures from 55-70.
There are several possible reasons why you might not be getting amplification of the combined product in your overlap extension PCR. Here are a few suggestions for troubleshooting:
I hope these suggestions help you troubleshoot your overlap extension PCR reaction.
Waiting for the answer also.
I tried with ~100ng of each fragment into a total of 20uL of PCR without primers. It works once with high-fidelity DNA polymerase, but when I repeated it, I get nothing.
Some say that needs to adjust to an equal molar ratio and the PCR condition for the first 10 cycles is Denaturing 20s, Annealing 20s and Extension 20s, then increase the condition according to normal PCR and primer can be added.
I am still working on it, if anyone has some tips and tricks to make it please share.
Thank you.
Hi,
I am having the same problem. Did you manage to solve it?
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