In article <2rafqt$gqt at mserv1.dl.ac.uk> you write:
>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
Are you using a probe labeled at different time than the one for library screening? One possiblity is that the vector instead of the insert for the probe was isolated from the gel and labeled. No insult, but this happens if one works in a hurry.
If you still have the hybridization solution (if it is <2 month old) which you used for the library screening, i would suggest to use that to hybridize with the plasmid blot. I like to reuse the hybridization solution. It always gives me cleaner signal, maybe the dirts has been absorbed when it was first used.
Hope this helps. Good luck.
: Xiaoying Lin <xlin at umbc.edu> Phone:410-455-2263 Fax: 410-455-3875 :
: Biol. Sci./U. of Md Baltimore County, Baltimore, MD 21228, USA :