Problems with lambda
eestraus at staff.uiuc.edu
Wed Apr 23 09:36:33 EST 1997
I am trying to make a cDNA library using Stratagene's lambda zap
express. I have been having a lot of trouble getting lambda to behave
itself. So far I don't have any pfu's in my library, but I have gotten
some of the controls to work very inconsistently. I want to try to get
the controls working better before I work more on the actual library.
My problem is that I can only get plaques to form about half the time I
plate things out. I either get close to the number of plaques expected
(using the ligation control from the kit) or none at all. Every time I
get a nice bacterial lawn (I don't have confluent lysis), but only
sometimes do I see plaques on it. I am using Xl1-blue MRF' for plating
and it comes from the same glycerol stock each time. I grow the cells to
OD600 of about 1 and then dilute in 10mM MgSO4 to OD ~0.5. I then add
phage and incubate at 37 for 15 min. I then add top agarose & pour onto
a plate, let it harden & incubate overnight at 37.
I have replaced the MgSO4 & had the new batch work. I have made new
plates, LB, & top agarose. I see nothing consistent with batches of
these reagents. I HAVE NOT yet replaced my chloroform. Maniatis mentions
that bad batches exist, but since things have worked sometimes I assumed
that was OK. I am going to use new chloroform next time.
I would appreciate any suggestions. I think I have tried everything,
but there must be something I missed....
Thanks for your help,
Dr. Ethan Strauss
Department of Natural Resources and Environmental Sciences
310 Madigan Building
University of Illinois
Urbana, IL 61801
More information about the Methods