HELP!: library amplification

Kevin O'Connell kfoconne at facstaff.wisc.edu
Fri Jun 1 12:39:25 EST 2001


Hi All,

I am hoping that someone will be able to help me with what should be a
simple procedure.  I have a 1.5 ml aliquot of an unamplified two-hybrid cDNA
phage library whose titer is purported to be 10e10 per ml.  However I have
titered it myself and found it to be 6x10e7.  The person who constructed it
claims that it has 10e7 independent clones.  I wish to amplify the library
and it seems to me that I would have to use the entire 1.5 ml aliquot or
else the amplified library would have far fewer independent clones than the
original.  The questions are: Can I or should I use the entire 1.5 ml and if
I do, what volume of a log stage bacterial culture would I need to use.
Also what is the maximal number of PFU that can be plated on a 150 mm plate.
That is how many plates would I need.  One protocol call for 10e5 per 150 mm
plate but if I used the entire aliquot I would have to use over 100 plates
and this is obviously way too many.  If anybody has some suggestions, I
would greatly appreciate it.

Kevin




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