Large Scale E. coli Transformation

Jeff Huckaby jeffrey.huckaby at yale.edu
Wed Apr 28 10:50:28 EST 1999


Netters
    I need a protocol or tips on doing a large scale ligations and E. coli.
transformations.

Here's my problem.
I have generated approximately 10^9 gene variants by mutagenic PCR.  First,
I have to clone these PCR frags into my plasmid and then get ready to
transform.  By my calculations, I will have to transform with well over a ug
of DNA to produce the number of transformants that I desire.  This problem
is similar to making a cDNA library but dealing with ~100 fold more
transformants.

Has anyone transformed with microgram quantities of DNA?

I know that the transformation efficiency drops dramatically when using
large qty of DNA in a small volume of cells (50-100 uL), but what if you do
transformation in 10mLs of competent cells?

Any tips on large scale transformations and/or ligations will be
appreciated.

Thanks
Jeff Huckaby M.Phil.,M.S.
Dept. of EEB
Yale University





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