minipreps from colonies

Michael Benedik bchs1b at Elroy.UH.EDU
Wed Aug 11 13:47:46 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


Are your plates old. If they are a little old much of the amp may be gone
and by the time you get fat colonies you are looking at cells with
no plasmid.


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 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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