I extracted plasmid from ETEC and positive control(DH5a), and there were no problem.
And then enzyme digestion. but I found that the positive control plasmid was digested perfectly, but I couldn't find any bands from ETEC plasmid. ( I used same DW, enzyme and all buffer)
To solve this problem, I did PCR and requested sequencing. and also, there were no problem.
But when I digest plasmid using enzyme, there are no bands everytime. (I think the ETEC has something like nuclease, so the plasmid had composed..maybe?)
Does anybody know the reason?
Dear Garam,
Even if the ETEC had such putative nucleases, they will be taken care of when you perform plasmid extraction. Have you checked the number of sites at which your restriction enzymes cut within your plasmid?
Abhishek
Similar problem occurred. Incubation of the plasmid only with NEB buffer at 37℃ is sufficient to result in smear.
PK treatment or Phenol-chloroform extraction can remove the nuclease contamination.
So your speculation does make sense~
If you have doubt about the presence of your ETEC plasmid as extraction product, you should try another restriction enzymes for which there are available sites.
Mostly the absence of reaction, if everything else is ok (plasmid, buffer etc.), is due to lack of available sites of cut.
Dear Garam wi
I my opinion, Expression bacteria does some modification in restriction sites of transformed plasmids, So you can not get any restricted product like cloning bacteria (Dh5a). you can check your product by colony PCR.
you can also check restriction digestion (same reaction mixture) with positive and negative control and with and without restriction digestion of ETEC and Dh5a extracted plasmids. Run the gel and see the differences between restriction. I hope you will get positive result and good conclusion.
Best Regards
Dr. SSY
Maybe, there exists a possibility that the plasmid extracted from ETEC accompanies some substance which can affect the specifity of restriction enzyme.
Dear All,
In my opinion, “but I couldn't find any bands from ETEC plasmid” meant that Garam wi didn’t find neither circular(uncut) nor linearized(cut) bands from the gel after restriction digestion. In another word, the plasmid was degraded in restriction digestion and became smear after running the gel. If so, which restriction site to choose may hardly matter at all. Just speculation. Garam wi will find the real answer. ^^
Best Regards
Peng
DH5a is an E.coli strain with several mutations that facilitate the plasmid cloning procedures. Here is the genotype of DH5a: F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1
In which endA1 means a mutant form of endonuclease A, so that plasmid DNA extracted from DH5a will not be degraded. But ETEC is a wildtype E.coli that contain functional endonuclease A enzyme and it may tagged along the plasmid DNA during extraction. In the presence of Mg2+ from digestion buffer, endonuclease A chew away your plasmid.
you need a good plasmid isolation kit that can remove endonuclease from your plasmid DNA.
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