lambda gt11 screening woes

mehra at ncl.ernet.in mehra at ncl.ernet.in
Mon Jul 4 02:42:18 EST 1994


Hi folks on the network!

I am a guy who has lost a lot of hair and sleep trying to
 pursue a doctoral degree.

I have a potyviral cDNA library in lambda gt11. This I
screened with an antibody and I got immunopositive
plaques. When I isolate the DNA from these plaques and
digest them with Eco RI, I don't see the insert. I know 
somebody who faced a similar problem, and he just changed 
his vector. I have come across articles where lambda gt11
libraries have been screened with nucleic acid probes, or
inserts have been amplified using PCR (I shall be going for
the latter strategy as soon as my oligonucleotide primers
are ready), but none of them say why (or whether) 
immunoscreening doesn't work. Promega, Stratagene,
Pharmacia et. al. market the product for immunoscreening,
so do Huynh, Young and Davis claim in their publicationó (PNAS
(1983© 80¬ 1194-1198» DNÁ Cloninç º Á Practicaì Approacè (IRL
Press¬ Oxford¬ D® Glover¬ Ed.© 49-78). Only
the New England Biolabs catalogue mentions a possible loss
or modification of the Eco RI sites, and suggests a Sac I-
Kpn I double digestion. These are sites flanking the Eco RI
cloning site. I have tried this, and also a  
digestion with Bgl I, But my gels show beautiful ladders
 everywhere.

Is there anyone there who has had a similar problem with
the same vector and has cracked it? 

Once I get the hallowed degree, I promise you a drink at the
nearest bar!

Thanks!!!!

Tomal
                                  Tomal K. Dattaroy
                                  Plant Tissue Culture Division
                                  National Chemical Laboratory
                                  Pune 411008.
                                  INDIA
                                  E-mail: ptc at ncl.ernet.in
 




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