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
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.