Gilbson ligation fidelity problem

Does any one have an opinion regarding the fidelity of Gibson ligation kit from NEB. We found there are about 30-40% of the constructs made from gibson ligation have some kind of mutation after deep sequencing, which is less likely from the synthesis error of the oligo pools. And almost all of the mutations are within the region that during gibson ligation will be subjected to chewing and re-synthesis. I wonder if this is because the polymerase in the gibson kit is low fidelity or because the oligos pools have the sgRNA secondary structure.


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Lemon Elfabout 2 years ago

I met the same problem as you described recently. I am wondering if you have solved this problem? If so, how did you solve it? Or did you directly use this library for downstream applications? Thank you!

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