In article <94128.110652MALBOOBI at QUCDN.QueensU.CA> <MALBOOBI at QUCDN.QueensU.CA> writes:
>Dear Netters:
> I am screening A. thaliana genomic library with a heterologous probe. By
>screening about
>160'000 plaques(!), I have got 9 independent clones. These clones are purified
>to homogeneity by 3-5
>time screening. All washes were to high stringency. To double check that these
>(or at least one of them)
>are corresponding to the right gene and not other related genes (i.e. other
>members of a gene family), I
>do the following test primarily. That is, I made a southern blot of A. thaliana
>genomic DNA digested by
>3 different restriction enzymes. Then, I hybridize the original probe (which was
>used for screening) and
>the probe made from the whole insert (9-17 Kb) of the isolated clones. I
>expected that the banding
>pattern of the S. blots of the isolated clones to be similar (or include) that
>of the S. blot probed with
>the screening probe. This did not happened for any of the isolated clones. Any
>advice or comment
>regarding this problem is greatly appreciated. Thanks. Ali
>My E-Mail Address is: malboobi at qucdn.queensu.ca
Ali,
If I understand your question correctly, you are using the same approach
we use in my lab to verify clones of genomic DNA. It is essential,
especially with inserts as big as yours, that the DNA for the genomic
digest is from the _same plant_ as the DNA used to construct the
library. Even that might not work if the gene you've cloned is highly
polymorphic. If the two DNA samples are from different individuals,
your chances of finding the same restriction patterns are reduced.
Good luck.
Joe Bagshaw
