To know the reason I kept separate reactions each with 1ug of plasmid and the reactions as follows.
1.Plasmid with nuclease free water
2.Plasmid+water+buffer
Incubated at 37c for 4hrs and rsults are as follows
Reaction 1 - Intact plasmid
Reaction 2 - Smear was observed
To overcome the chances of nuclease contamination in buffer I kept a separate reaction with new buffer for double digestion and changed restriction enzymes from fermentas to neb, still I have observed smear on the gel.
what kind of competent cell you use ? some competent cell like stbl3 from invitrogen will be left over some endonuclease. In my experience, i used traditional phenol-chloroform method to extract my plasmid DNA, and no smear was observed after digestion. You may also choose some plasmid extraction kit with endonuclease remove solution. Hope this would help you ! All the best.
To me it seems that the source of nuclease contamination is your plasmid, as there is degradation even after changing buffers, enzymes, etc. (don't be misled by the fact that incubation in water does nothing; many nucleases need the divalent cations, pH, etc. provided by the buffer). I'm assuming of course that the nuclease-free water is indeed nuclease-free.
What strain and DNA purification method did you use? Use a silica- or IEX-based cleanup kit to further purify the DNA. I once had a plasmid prep from a dam- strain that degraded upon incubation at 50*C for a Bcl I digest, but not at 37*C (go figure). The nuclease would go away by clean up on silica-based columns, but was resistant to extraction with phenol or phenol/CHCl3.
Thanks for ur suggestions Yang and Martin....
I have used thermo plasmid mini prep kit for plasmid isolation.
Hi!just want to follow up this issue, do you manage to solve this issue?
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