While I was amplifying a certain gene from chromosomal DNA for my Gibson Cloning, I noticed after sequencing that there is one extra A on the middle of my gene of interest sequence. In the original sequence there are six A (AAAAAA) but when I cloned on the plasmid, the whole plasmid sequencing showed seven A (AAAAAAA) making a frame shift mutation. I used Hight Fidelity Phusion DNA polymerase for PCR amplification. Do you recommend any other polymerase?
Thank you so much for your suggestion.
Hello,
When you sequence, do it for 4 or 5 amplification products and get the correct one without mutation.
For polymerases with fidelity, there is Taq Gold DNA polymerase.
Also, tried to reduce the number of PCR cycles, to reduce the possibility of mutations appearing.
High Fidelity Phusion is very high fidelity, I don't think changing polymerases would be that useful. I would agree with Sofiane Benyamina that you should just isolate a few independent clones and sequence them to find one that is correct before trying to change things.
I agree that reducing the number of PCR cycles and sequencing more clones should work. It could also be a sequencing artifact, see if you can get forward and reverse read coverage for that area and if the extra A shows up in both or not.
I agree with Michael J. Benedik, I think no need to change the POLYMERASE. I have experienced of good performance of Phusion high fidelity mastermix (NEB). I hope , you have added DMSO in reaction, if not then you can perform PCR with DMSO as additives @5-10 %.
Thanks a lot Aman Kumar , Amy Klocko , Michael J. Benedik and Sofiane Benyamina for great ideas! Finally I got the right insert in the plasmid! I did PCR again with 3% DMSO with freshly isolated template DNA. Then Re-did the Gibson assembly. Before isolating plasmid I did a colony PCR. The colony that gave me expected band were selected to make overnight and for plasmid isolation. Then I send couple plasmids for sequencing. I got the result back today and it works for both !!! Thank you so much for your help!
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH