Recently, we observed with one particular construct consistently single bp deletions in Gibson assemblies. The 1bp deletions almost exclusively occur in the areas of the primers used to amplify the products, but are not always the same. We have redesigned and reordered primers, but this did not make a difference. We have changed E.coli cloning strains, no difference. We have changed the time of the assembly, no difference. Any suggestions as to why this could be and how we could tackle this?
If the deletions only occur in the region of the primers and are not consistent then it really would suggest poor QC by the manufacturer. Order primers with either PAGE or HPLC purification to ensure you only have oligos of the required sequence, they are a little more costly but not as expensive as your time!
Agreed. I would run them through a 7M UREA, PAGE gel if they are in the 20-60 bp rage, you can separate single base deletion/truncation products.
Hi. what polimerase are you using? phusion could be a good idea to use
Hi, our long primers are generally RP purified, not HPLC or PAGE. I will try this out next time. We usually use Q5 (NEB) with - so far - excellent results. I will give Phusion a go, but dont think it is a polymerase issue as it occurs primarily at the site of the oligo. Thanks!
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