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
> I have just been told that another group here has had the same problem
> they too have no idea what is going on.
I have not had this particular problem, but a similar one. A while back I
was trying to do some cloning in Bluescript and found that I frequently got
anomolous sequence data from my clones ("DNA from space" as I call it).
It turns out that Bluescript has a high frequency of mutations. This
mutation frequency is strongly enhanced by storing E. coli containing the
plasmid on plates in the fridge for a couple weeks. The mutations frequently
included new DNA (always some in the size of the fragment I was trying to
clone) with no homology to either Bluescript or my insert. They also
included loss of a few restriction sites, or the ability to make ssDNA, with
no apparent change in size. I asked around on the materials and methods net
at the time, and found that many other people had experienced similar
problems. It is not exclusive to Bluescript, but this plasmid does seem
particularly prone to these strange mutations.
My imediate solution was to duplicate all the mysterious clonings in pTZ
(where they worked like a charm), and to avoid strains that have been stored
on plates in the fridge. Storing strain stocks as dry DNA, or as glycerol
stocks, or as lyophylised cells under nitrogen are much more reliable.
Leonard N. Bloksberg
bloksber at pilot.msu.edu
Dept. of Crop and Soil Science
Michigan State University