发信人: yuffie (天天想你), 信区: Biology
标 题: Re: cloning help!
发信站: The unknown SPACE (Tue Oct 8 14:26:48 2002) WWW-POST

If contamination goes 99.9% in all clone, that means you get a supercoiled
circular DNA as your contaminant. It transforms at a much higher efficiency.
First thing you might do is change plasmid.
I talked it to my boss, he had an interesting theory. If your insert confers
toxicity to E.coli, he said there might be some wierd recombination event that
cut the insert out of the plasmid, along with flanking reagions (which might
contain the MCS, that is why you cannot cut it again with your REs). Circular
DNA runs differently from linear DNA, so 13kb might look like a 8k, but it
will be a smear instead of a clear band. Gel purification cannot get rid of
it, and supercoiled DNA just transformed a lot better. I do not know if there
is a way to get rid of those supercoils, maybe Topoisomerase?
If that indeed the case, it will be difficult to deal with. Because any
vector you put it in, it might happen again.
If the contaminant is come other plasmid, you can try cut your mini-prep with
some common RE which have no cutting site in your plasmid, hoping they will
cut the contaminant into linear, then transform E.coli and mini-prep, RE
digestion for correct pattern. Or, if you can get enough 8.6k piece to
subclone into another vector which has different antibiotic resistance.
If you don't have enough, then find a single cutter inside your insert, treat
your plasmid with it, it should linearize it at 22k linear DNA. Gel purify
it, it should be different size from those circular DNA. Ligate it with
ligase, make it circular again, then transform E.coli.

--
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