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minipreps from colonies

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Tue Aug 10 17:42:58 EST 1993


In article <1993Aug10.093228.2704 at gnv.ifas.ufl.edu>
 afc at gnv.ifas.ufl.edu writes:

> We have been working with libraries in Promega's phagemid lambdaGEM-4
> vector.  After popping out the insert + plasmid (pGEM-1) we transform
> into DH5alpha, plate on ampicillin, and pick a single colony.  We then
> restreak this and have our subclone.  This works great.
>
> However, if we try to scrape some cells off of the restreaked plate to 
> do a miniprep, we get no DNA.  About ten clones of different genes from
> different species, using either alkaline lysis or boiling, have failed.
> If we grow the cells in broth, no problem, lots of DNA.  I have been doing
> minipreps off of plates for ten years and never had any problems before 
> this.  
>
> This is not a major problem, but it is one for which I cannot come up with
> a logical explanation.  I presume that it has something to do with the
> vector, the host, or some interaction between the two.  Anybody know the
> answer?
>
> Andrew Cockburn

If the plates with DH5alpha colonies are old or stored at 4 C, the cells
produce a fair amount exopolysaccharides which might interfere with your lysis
procedure.  Maybe you could try resuspending the colonies in 1 M NaCl as per
the mini-prep method I've posted previously. Also, I find that the hot alkaline
method helps the lysis of this strain. I think you would have no problem with
isolating the plasmids from colonies this way.

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA



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