I'm using a multistep approach to insert multiple genes in sequence into the pHIV lentiviral vector. I am PCR amplifying the genes from either gblocks from IDT or other plasmids. I use PCR to amplify the genes of interest, confirm their size by agarose gel, and then attempt to assemble them using the NEB HiFi kit (specifically the 2x NEB HiFi master mix). When I submit the assembled plasmid for sequencing I am able to confirm insertion of the gene of interest for that round of cloning. However, the problem I am experiencing is that after several rounds of inserting each gene, I have gone back and attempted to sequence the entire assembled cassette and am noticing there are random sections of gene sequences missing.
Has anyone else seen this? Does anyone have any suggestions for working around it? Is there a better kit available?
Hi Nicholas,
Never had problems with NEB HiFi. The source of your mutations is most likely PCRs, oligos or gblocks. Using Sanger sequencing results, determine where mutations tend to occur.
If it's in the overlapping regions of your assembly, this is most likely due to poor oligo quality. If it's within the fragments it can be due to poor PCR fidelity, use a high-fidelity polymerase (I'm using Phusion or Herculase II).
If you are already using a high fidelity polymerase and still observe a high rate of unexpected deletions/mutations outside of the oligo regions, these may already be present in your template gblock DNA.
Check what is the expected error rate for IDT gblocks, when resorting to synthetic fragments I'm using Twist gene fragments which have a error rate of 1:4000 bases. Last time I checked this was the lowest error rate for synthetic gene fragments on the market, and they were also much cheaper (0.07 $c per base).
Hope it helps
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