Plasmid Deletions?

R. John Lye rjl6n at uva.pcmail.virginia.edu
Mon Apr 6 18:32:27 EST 1998


Ed Dunphy wrote:
> My plasmid preps (Maniatis alkaline lysis) keep producing a secondary
> plasmid band on our gels that may represent a deletion product.  It's only
> 1% of the amount of the supercoiled and runs about 200 bp faster than the
> supercoiled band.  

Yeah, I've seen that on plasmid preps.  It's an alternate
form of your plasmid (form 4, is what I think its called).
It doesn't cut with restriction enzymes either.  Its produced,
in my hands anyway, by leaving the prep in the lysis stage
for too long.  I limit that step to no more than four minutes
at the max.

> I've even cut out the supercoiled band from the gel and
> retransformed it with the same results.

Yup, because its still your plasmid and is caused by your
preparation method.
 
> What the heck's going on and how do I cure it?

Be careful to limit the amount of time in the alkaline/SDS
stage.

good luck,



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