Hello. We are trying to clone a2-3 kb long insert into a 3,5 kb long vector by NEBuilder. We had to amplificate by PCR both the insert and the vector. We used MyFi polimerase.( https://www.bioline.com/de/myfi-dna-polymerase.html ). The products contain 2-3 mutations, so the polymerase made mistakes, but it is a proofreading, high fidelity taq... Why could it happen? What circumstances could cause this?
It might not be errors from your polymerase. It's possible that your DNA template has SNPs compared to the reference genome.
Any polymerase can made mistakes! Also, 2-3 mutations are not out of the range in 2-3 kb long!
3 errors in 3 kb would be very high for a proof-reading polymerase. Another possibility is that you are not seeing errors in the PCR, but instead uncertainty in your sequence files. Do your chromatogram peaks look distinct with high quality scores?
If nothing else, you might just have to sequence more inserts until you find one that doesn't have any errors.
Thank you, for your answer. Unfortunately sequenograms look good, and we verified it from reverse direction as well. So, errors are in the PCR, and I also think this is too much error for a proof-reading taq... :(
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