Hirt extract purification??

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Thu Dec 5 22:20:32 EST 1996


In article <587q7m$6iqm at piglet.cc.uic.edu>, levenson at uic.edu (Victor
Levenson) wrote:

<snip>

Only 10-100 cells??? Even if the amplification of the vector in the COS
cells results in 1000 copies per cell then your 100 cells would only have
100,000 DNA copies to transform your E. coli - roughly 2 X 10^-19 moles of
plasmid (for a 5 kb plasmid roughly 0.66 pg)....and this is assuming 100%
recovery from the Hirt.

I would suggest you "dope" lysates of untransfected cells with an
appropriate series of plasmid amounts, continue the processing of the Hirt
extract (precipitation and washes) and then transform your DH5a. If the
colony numbers jibe with the predicted number of colonies based on
absolute recovery and perfect transformation then the DNA extraction is
likely not the problem.

Cheers

Karl the hepB guy

-- 
Karl Fischer
tyr-2 at bones.biochem.ualberta.ca





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