Death of a lambda library

Tae Shin tshin at husc8.harvard.edu
Mon Mar 22 00:53:42 EST 1993


Hi.  I've been working with a lambda ZAP library from Stratagene for the past
few months in the process of expression cloning.  About a month ago, the
library's titer dropped precipitously, the plaque size shrank, and now the 
library appears to be completely dead.  I was using BB4 grown in 0.2%maltose,
10mM MgSO4, 10ug/mL tetracyclin.  This was diluted 2x in 10mM MgCl2, 10mM
CaCl2, and 600uL was infected with 60000 pfu of phage.  This was plated on LB/
Tet/Agar plates in 0.75% top agarose.  These were incubated for 4-5hrs at
42deg., then overlaid with Immobilon HATF filters soaked in 10mM IPTG. 
Incubation at 37deg for 4-6hrs usually would give the expected number of
plaques, which were small but visible and distinct.

I don't expect anyone to solve my problem, but in general does anyone know
of factors that can kill phage or inhibit its growth?  I've remade all of
my buffers and gotten fresh bacteria, but the results are the same.  Any
thoughts would be appreciated.

Thanks,
T.B. Shin
tshin at husc8.harvard.edu



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