Problem on transformation

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Fri Nov 20 15:09:17 EST 1998


In article <36565F4A.3D6EA674 at itsa.ucsf.edu>, longli at itsa.ucsf.edu wrote:

> Hi, Everybody:
> Recently I got a strange problem trying to clone a promoter sequence
> into pGL3 enhancer. After transformation, we could get positive  clone
> as tested by PCR of the minipreps. The problem was the positive clone
> became negative after maxiprep either tested by PCR or digestion. This
> phenomona repeated itself several times. If anybody has any suggestion,
> please give me a reply. Thanks.

1) Did you check the minipreps by restriction analysis after you
had performed the preliminary test by PCR?

- Just worried that your PCR result was from amplification of
a very minor population that was eventually diluted and lost.
A quick digest should allow you to see if you "really" have
the plasmid.

2) Does your insert have a lot of direct or inverted repeats?

- These can act in an unstable manner in some host strains.
You may want to consider switching to HB101 or SURE which are
reputed to reduce the incidence of rearrangement.

3) Is there any chance that your insert is toxic?

- You could try growing your maxiprep at lower temperatures,
for shorter periods of time and/or in a richer medium or in
one of the strains above (there are others).

I assume you need lots of plasmid so I hesitate to suggest
that you generate the quanitities you need by lots and lots
of minipreps (how much do you *really* want that plasmid?).

     Good luck,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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