Dear yeast people,
I am doing a two hybrid screen and I get a lot of times the same cDNA,
probably a very abundant one. I would like to screen my positives (about
100 colonies) against this cDNA using colony DNA hybridization on nylon
filters. I used this method with bacterial cells and works nicely, but I
know that for yeast is more difficult because it is hard to break the
cell wall. I already tried once, following a protocol I found in the
clontech "yeast protocols handbook". In this protocol, cell walls are
disrupted first by incubation for several hours in sorbitol/
beta-glucuronidase. It did not work for me, the background was extremely
high.
I wonder if you know of a good protocol for yeast colony hybridization,
if such a thing is possible.
Thanks a lot for your help.
Joaquim Culi
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