Hey there,
So I did a Gibson assembly and transformation yesterday and today I saw colonies! I will make a liquid broth and extract DNA to confirm next week, but until then here’s a question:
2 of the 6 fragments in this assembly were genes amplified off of a plasmid. I used the PCR products directly in Gibson assembly which by my calculations would have carried over around 2-5 ng of plasmid template DNA to the gibson mix. Is there a solid chance that my plasmid dna survived the PCR thermocycling to confound my results? Or would the cycling have reduced the amount of intact, transformable plasmid DNA?
Any guidance would be appreciated!
Plasmid in PCR would lose supercoiling which may affect its transformation efficiency. But it can still transform E.coli colonies. Anyway, why are you not using DpnI enzyme to destroy the plasmid DNA after PCR? This is a common procedure to degrade methylated plasmid DNA that comes from E.coli lab cells.
Yes, there is a good probability for the presence of parent plasmid DNA after PCR.
Plasmid DNA can and will , definitely survive thermocycling. That's why for example in SDM (site directed mutagenesis you use DpnI to get rid of the parental plasmid).
A picture of plasmid DNA after Thermocycling. Just 1 µl of plasmid DNA was loaded. You can see the clear band. Cooper A. Svajda
Probably too late but if you would like an alternative to clone 6 fragments I use this protocol. doi: https://doi.org/10.1101/2021.09.04.458594.
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