---------- Forwarded message ----------
Date: Thu, 19 May 1994 15:03:54 -0600 (MDT)
From: Hiranya Roychowdhury <hroychow at nmsu.edu>
To: "Leonard N. Bloksberg" <bloksber at pilot.msu.edu>
Cc: arabidopsis at net.bio.net
Subject: Re: bluescript
On Thu, 19 May 1994, Leonard N. Bloksberg wrote:
> In Article <2rafqt$gqt at mserv1.dl.ac.uk> "Neil Butt <N.J.Butt at sussex.ac.uk>" says:
> > Dear Netters,
> > We have recently been screening ZAP libraries and getting what appears
> > to be positive signals. Primary screens give reasonable duplicates and
> > secondary screens show strongly reacting plaques. However, after in vivo
> > excision and purification of the resulting bluescript plasmids the
> > problems begin. We can excise the cloned inserts and blot these to
> > confirm the reactivity of the inserts. What happens then is odd, we seem
> > to get very strong hybridisation to the vector and very little to the
> > inserts. This has occured after very stringent washes (0.1 x SSC 65 C
> > for 30min, 0.1 x SSC 30min 65 C, and 0.1 x SSC + 0.5% SDS 30 min 65 C).
> > Database searches show our probe has no homology to the bluescript
> > sequence, so why should this happen? If it was specifically reacting to
> > bluescript you would expect everything to hybridise, but this does not
> > happen. I've become rather desparate in searching for a solution so I
> > hope someone can help.
> > Anxiously awaiting a reply
> > Neil Butt
> > Department of Biochemistry
> > Univeristy of Sussex
> > Brighton, BN1 9QG
> > U.K
> > P.S.
> > I have just been told that another group here has had the same problem
> > they too have no idea what is going on.
I saw two postings about the above paradox. I am having the same problem
with ZAP libraries. After screening the libraries with the relevant probes
and picking the plaques that showed strong signals (sometimes after a
quaternary screening), I prepared phage DNA, cut it with RE and did
Southerns. Upon hybridization at moderate to high stringency the lambda
arms lit up with no signal at the insert. In order to eliminate the
possibility that there could be carry over from the plasmid vector in the
probe, I selected a 0.5kb piece from the middle of the probe DNA. Same result!
There were reports in the 70's that some plant DNA hybridize rather
strongly to pBR322 sequences. I wonder if that has something to do with it.
It will be interesting to see whether those of us that are faced with this
odd situation with ZAP are working with a set of genes that have something
in common. Because, not every plant molecular biologist have come across
this phenomenon with ZAP and there are a lot of ZAP-users. If it has
something to do with the pBluscript sequences, then other blue-white
selection systems (eg. gt11) may pose similar impasse.
We will definitely appreciate any helpful discussion on the subject.