Hi,
I have to cut some plasmids (natural plasmids)in order to determine the size on gel electrophoresis. I would like that the enzyme cuts the plasmid once, but I don't know the sequence of plasmids, so I don't know how to choose the suitable enzyme for this digestion. Can use suggest me what I can do?
Thanks
I would do a series of single enzyme digestions with all restriction enzymes you can find in your lab. If you get a single cut (linearized) the plasmid will run slightly larger than the uncut (supercoiled) therefore always have a no enzyme control. It should be visible on the gel if the enzyme cuts more than once unless the distance between the cutting sites is small. But since you can get only somewhat rough size estimation from the gel two restriction sites in close distance won't affect the outcome.
You could also google for restriction site distribution in your bug if somebody already some research. Wouldn't give to much on these results.
Good Luck
Cornelia is correct, you need to identify a suitable enzyme by trial and error and test a number of different ones. Remember that you should probably limit your analysis to those enzymes with 6 bp or longer recognition sites, the 4 and 5 base recognition sites are going to all give you multiple cuts.
Yep, you are going to have to do this empirically. I'd try the common and cheap ones first (EcoRI, BamHIII, XbalI) and hope to get lucky. If those cut too often, you can go for the enzymes with larger recognition sites. Good luck!
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