Hello,
I am trying to figure out the issue with the isoform of pDNA isolated with GeneJETPlasmid Miniprep Kit and further digested with FastDigest enzyme in FastDigest Buffer (thermo scientific). As a control for restriction digest I prepared the exactly same reaction with pDNA, but didn't add the enzyme. I loaded on 1% agarose: marker, pDNA in restriction digest mix without enzyme, pDNA in restriction digest mix with the enzyme (see the pic attached).
The enzyme recognizes a single site, so what I expected to see on the gel was the supercoiled plasmid DNA band in the middle lane, and in the right lane, digested, linear pDNA band, at slightly higher position than the middle one.
Instead, the middle one is a smear, that can hardly migrate through gel and I don't see pDNA at all. I don't think that it's genomic DNA or RNA either (used SYBRsafe for staining), as the right lane is a clear band which also corresponds with the expected pDNA size.
My question is, could FastDigest Buffer disrupt the supercoiled pDNA into open circular or nicked isoform? Currently, I cannot perform gel electrophoresis, with pDNA alone thus I hope someone has some more experience in this matter.
Thank you
Hi there,
I would suggest to re run these 2 samples again along with the starting plasmid.
It might be that your plasmid is a dimer (or more) and it gets converted to its monomer size upon digestion. If you are using a RecA- strain then whatever multimeric state the plasmid is in will be locked in place.
Yes, it is possible to disrupt supercoiled pDNA after isolation from E.coli with restriction digest buffer. Restriction enzymes recognize specific sequences of DNA and can be used to cut DNA molecules at these sites. The restriction digest buffer contains the necessary components to ensure the activity of the restriction enzymes.
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