Hi everyone,
I am trying to remove approximately 70 bp from a gene. Those bp correspond to the N terminal of a protein and I want to get rid of it. I have designed 4 primers so that I get my gene in two different pieces -without the N terminal- and then glue them together.
I cloned the gene and send it for sequencing and it appears that ONE base is missing (in the area that the internal primers are) so the protein is not in frame and I can't use it.
I will try to remake the gene by PCR. Do you have any suggestions for optimization? Should I use TD PCR? Is there a possibility the mistake is not due to the PCR process in general?
I used Phusion, I tried gradient PCR to compare yields of product and the primers Tm are 56-58 oC.
Thank you in advance
Did you sequence more than one clone? The PCR enzymes can sometimes make mistakes or edit the primer sequences. Limiting the number of cycles is helpful and trying other enzymes such as KOD HotStart or Platinum Pfx. Phusion should be good too but each enzyme has its own quirks. Lastly, if you're willing to spend for another primer pair, there are many PCR based methods for performing in-frame deletions, such as inverse PCR + ligation or site-directed mutagenesis by PCR. The attached paper is old and there are many variations on this theme now but this method worked well to delete or insert sizable fragments from ORFs in order to modify the encoded protein. I think in your case I would try inverse PCR and then sequence a few resulting clones. Good luck!
Possibly, it's a matter of cloning. As DNA is degraded by exonucleases, only one mising base at the end is quite a good result, I mostly lost up to one dozent nt's from the end of a 4kb fragent (cloned in a vector, amplified by phusion). sequence other clones or amplify and clone again. With some luck, next time the ligase is faster than the exonuclease. cheers
It can't be a matter of cloning since the missing base is not at the edges of my gene. It is in the middle, around the area the N-terminal was. Let's hope it's a random mistake of the polymerase. I am trying to remake the gene and I will send it for sequencing before cloning. Thanks!
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