transformation

Nicolas Kuperwasser nkuperw at yahoo.com
Fri Feb 8 20:05:52 EST 2002


Kyle Legate <legatek at mcmail.cis.mcmaster.ca> wrote in message news:<Pine.SOL.4.33.0202081143030.542-100000 at mcmail.cis.mcmaster.ca>...
> 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?t 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.
> 
> ... . . .  .  .  .    .    .    .     .     .     .      .      .      .
> legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com

Potenial reason #3...your enzyme(s) is/are bad.  May want to reorder new enzyme.

nick




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