I have been using Gibson Assembly to insert a 7kB fragment into an 8kB linearized vector. After assembly many colonies form, but colony PCR on the transformation consistently shows a 1.5 kB product instead of our expected 7 kB. RE digest of the plasmid shows a single band at ~10kB. There were no off target bands of the PCR fragment which contains 25bp of overlap with the vector and GC content of ~60%. The vector was dephosphorylated during digestion to prevent re-ligation.
What might this insert be?
I am using a stable E.Coli strain, but could recombination explain this?
This is weird! It appears to me that the problem is not really with the technique; it is most likely that your prime pair is not very specific to the fragment. Probably your primers target a different sequence instead of your fragment. You can recheck or redesign the primers.
Dear Andrew,
it seems quite weird, since the size of the construct you are getting is not matching with any combination. I would recommend to redesign the primers and check carefully the non specific pcr products. if possible you can gel extract both the fragments. Though recombination can explain your findings but then you have to match the sequence in the construct with the host genome.
Dear Andrew,
Is it possible that your 7 kb PCR never worked and you instead isolated the template plasmid and transformed it?
Does the restriction pattern correspond to the template plasmid of the 7 kb PCR? It should have the same selection marker of the final product to in this case.
Hope it helps
Francesco
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