I am trying to replace a 20 bp region with a different sequence of same lengthin a 10kb plasmid. I designed overlapping primers. However after PCR I am only getting the template. At first I thought I was getting amplification but on Dpn1 digest everything is getting cleaned up. I tried both multiple ramp and single ramp. Did multiple temperature gradients. But always the same issue.
You can try Gibson for mutagenesis. You can find the protocol online. Just need to design your primers again and perform a PCR to amplify the short fragments. Using HiFi DNA assembly master mix, just joint your fragments and transform directly. You will definitely get your mutant.
I don't recommend using Gibson (or similar kits) to assemble your plasmid using 20bp fragments. The 20bp fragment would be chewed up by the exonuclease in the reaction mix.
I would triple check your primers. Make sure the correct DNA sequence is in your primer. I would also recommend reordering the primer.
Since you are getting product (the 10kbp product), what exactly are you doing after the pcr reaction? If dpnI if chewing everything up, then you probbaly way too much DNA template in your reaction. I would drop the template 10-100 times and use excess primer. DM me the details and I can try to help. You need to further optimize your pcr and work-up conditions
I found the problem. The primer had a non specific binding site and that was causing a world of problems. thanks everyone.
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