I used the Gibson assembly to join together two PCR fragments (4400 bp +770 bp). After transformation of E. coli with the Gibson mix, I isolated DNA from single colonies and I performed a diagnostic digestion of every sample to select candidates to send for sequencing. I sequenced 9 samples in total and 8 of them had at least one point mutation/deletion. Only one sample was completely right in the insert region, but had some point mutations in other regions of the plasmid (sequenced later).
I knew that this is the downside of Gibson, but I expected to see polymerase mistakes less frequently. How often does a polymerase normally make a mistake? Can factors such as brand/quality of the polymerase (I used Phusion), PCR cycling conditions, particular regions of the double strand (GC-rich?) influence the performance of the polymerase?
This is not supposed to happen. DNA sequencing generates far more errors than a high fidelity polymerase. First, sequence them again, from both directions, and see if those "mutations" really stay in the same spots. Also look at the traces. See any funny jitters near the "mutations"?
https://www.nature.com/scitable/topicpage/dna-replication-and-causes-of-mutation-409/
polymerases vary with respect to error rates but the error rates quoted are for the enzyme working at its optimum temperature and buffer conditions. Most enzymes will make more errors at sub optimal ( low) temperatures so if the primers anneal at a low temperature in generating a product the enzyme still has activity at the lower temperature but will make more errors at the ends of the product
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