We have tried error-prone PCR with GeneMorph II Random Mutagenesis Kit and another kit from a Chinese supplier. Both kits worked fine in producing a specific product (single band on gel) of expected size. As a next step, we need to re-amplify for large scale cloning and to use the first round PCR product for second round error-prone PCR. This is where the trouble started.
Second round PCR only produced smear when we used the same primers and same PCR conditions. We also tried different Taq (Wolact), different template dilutions (100x all the way to 1000,000x), and different annealing temperature; all produced different degrees of smear. As a control, we used Wolact Taq to do the same PCR and use that PCR product as template for second round PCR; no problem was found in producing a single and strong band.
Why the error-prone PCR product is recalcitrant for second round PCR? What can we do to get around the problem?
Hi Nanfei,
We usually gel purify the band after error-prone PCR to insure that non-specific products are excluded in further steps. Traces of ethanol used in some of these gel extraction kits can lead to unproductive PCR as most polymerase are sensitive to traces of it.
If you want to avoid the gel pufification, there are PCR clean up kits or ethanol precipitation that can remove PCR reagents that could interfere with second round of amplification.
Good luck!
i also used the same kit to do the random mutagenesis, but i donot know why you did two rounds pcr, i always used the same template and it worked very well, on the other hand, if the mutation rate is high enough in the first round, probably the primer binding sites also get changed, that means in the second round the primers can not specifically bind to the target sites. so my suggestion is that, use the same template all the time, if your template was the genomic DNA, you should ligate the target DNA fragment to a plasmid as template. or maybe you can do two rounds pcr, but for the first round, you should use the fidelity enzyme.
good luck!
Hi,
Have you solved the problem yet? I got similar result after re-amplify.
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