Plasmid Deletions?

Claudia Sutton cas9 at cornell.edu
Thu Apr 9 03:01:59 EST 1998


In article <3529660B.4396 at uva.pcmail.virginia.edu>, rjl6n at virginia.edu wrote:

> 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.
>[snip]

Alternatively, and to check that this is what is going on, try doing
alkaline lysis with 0.15M NaOH instead of 0.2M. Works fine for me, and I
doing get the denatured form. 

Claudia (who, with help from other Meth-reag people, figured this out
several years ago)



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