I am doing site-directed mutagenesis using Inverse-PCR method to mutate a 10bp region of my insert. My vector plus insert size is 5.6kb. I am using Pfu polymerase from NEB to amplify my plasmid using mutagenesis specific primers.But, every time I notice bands around 3kb bp and 10kb , which is similar to the non-digested plasmid band. There is no specific band around 5-6kb in agarose gel. I have also just directly transformed my PCR product into the bacteria after DpnI digestion but that also did not give any colony. I have set a gradient from 57 to 66 C but, every temp. have shown the same result. The condition of the phusion PCR is-
1) Initial denaturation at 98C- 30sec
2) Then, 98 for 10 sec, annealing temperature for 30 sec, extension for 5 min ( each cycle) at 72C and final extension for 10min.
Any suggestion regarding this problem would be welcomed and appreciated .
Mayank,
I suggest you to do some following troubleshoot before you are doing your expermient as:
1. Check your plasmid stock and confirm it with multiple digestion.
2. Either redesign your primers with more complementary part sequence which ensure specificity of binding.
3. Try a different like kapa or phire like or hotstart enzymes for amplification.
I am not that familiar with Inverse PCR method but I agree with Koushik. You should check your template and test your primers.
"I have also just directly transformed my PCR product into the bacteria after DpnI digestion but that also did not give any colony. " - it clearly indicates that your PCR did not work or the amplified PCR product did not contain the correct vector sequence which is necessary for the cloning by bacteria.
I personally use whole plasmid PCR method and I have had issues with the wrong annealing temperature. Therefore, I have altered the annealing temperature and sometimes even redesigned primers. If you are sure that your template is correct, you should optimize the PCR conditions or redesign primers.
In addition to the above suggestions, if you get no colonies it may be that your transformation efficiency is not sufficient. If you were using Ca2+ home-made competent cells , you may consider switching to commercial competent cells or to electroporation to boost the yield of transformed colonies
Hi Maynak ,
I would encourage you to use the Q5® Site-Directed Mutagenesis Kit (NEB) for your next trials. I've used it several times in the last few months and it really works fine. In my case the amplified plasmids can be seen very well and the efficiency of positive colonies was quite high. It is worth trying it if you continue having problems. Good luck
It seems you are using mega primers for that you kept annealing temp very high. It is ok. I think you should keep extension at 70 or 72degree celcius for 12 min. Pfu amplify @ ~0.5 kb/min. Try to decrease the parent plasmid by 2-5 fold.
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