I build a big plasmid, more than 17kb， the sequence results are right, but the restriction enzyme digestion has some problems? single enzyme digestion should contain one band, but the results show two, one friend tell me big plasmid usually has problems with digestion. do any people know why big plasmid digestion has problems?
Two possible explanations:
1. Your plasmid does have a second restriction site. This may have been overlooked in the construction of the plasmid, or it may have arisen from spontaneous mutation.
2. Star activity of the restriction enzyme. This known to occur with enzymes like EcoRI or BamHI under conditions like low salt or high glycerol concentrations. However, in your case, this seems rather unlikely because star activity would be expected to generate more than one extra cleavage.
I do'nt believe that the extra cleavage has anything to do with the size of the plasmid
Also, plasmids can form secondary structures & will sometimes show up as multiple bands even if they are not cut.
Or you are seeing a partial digest & have linear vs circular plasmid.
What do you see when you run a sample of undigested plasmid along with your digested plasmid?
I had a few questions that might help identify the problem. Could you post a picture of the bands from the DNA digestion? If not, could you describe the approximate sizes of the two bands?
When you say "the sequence results are right," did you sequence the entire 17kb?
since your plasmid size is big, sometime one restriction site could present many times. so you have to consider the restriction sites at the plasmid backbone. I supposed some of the unique restriction sites at the expression cassette are present at the plasmid backbone sequence.
before carrying out the plasmid digestion you have to explore the sites number of the used restriction enzyme within the whole plasmid sequence. of course handling the big size plasmid is difficult in compare with the small size.
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