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minipreps from colonies

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Tue Aug 10 09:32:28 EST 1993


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



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