Kyle Legate legatek at
Fri Feb 8 11:49:06 EST 2002

On 8 Feb 2002, SHAHRAM NAZARIAN wrote:

> I have not been able to get the correct clone from the transformation. I
> extracted DNA plasmid but when I digest I don=B4t get any desired fragmen=
t. Do
> I have to linearize befores the restriction assay?
There are many reasons why a plasmid won't cut when you isolate it. Two
reasons off the top of my head are:
1. Dirty prep. If there's any phenol or salts present in your plasmid
solution it could kill/inhibit the enzyme. Solution: Ethanol precipitate
your plasmid again, and resuspend it in TE or water.
2. It's not your plasmid. If the strain you're using doesn't like your
plasmid it can rearrange it or mix and match your plasmid with pieces of
the E.coli genome. We've seen this even in SURE cells, which are not
supposed to recombine. Such isolated fragments frequently won't cut with
anything. Solution: grow your plasmid in a different E.coli strain.

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