genomic DNA in plasmid prep

Jim Graham graham at biodec.wustl.edu_nospammmmmmm
Wed Jul 22 05:50:03 EST 1998


> letting it sit too long in the alkaline lysis buffer

Among a miasma of possibilities. I think this now popular explanation is
overated. Try using a rich buffered media and growing the cells only a
specfic length of time at a well-defined anitbiotic concentration so
that they are just late-log at harvest. Then wash the pellet with 0.5 M
NaCl to remove extracellular material for lysed cells. Gently extract,
and do a hand shaken chloroform extraction on the cleared alkaline
lysate prior to ammonium acetate isopropanol precipitation (0.6 vol
alcohol and spin only 5 minutes without waiting). 

This may not work on Sundays or Tuesdays or whenever your time is most
pressed. :) I've yet to find any way of ridding samples of chromosomal
once its in the prep. (Tried some acid phenol, which can work if the
plasmid is not also removed.)

If you can solve this, you will be a hero. (Plese E-mail me if you have
a clue).



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