[Yeast] Yeast Yeast Colony Hybridization protocol-request

Wei Zhou zhou.wei at cmm.ki.se
Thu Feb 23 03:28:02 EST 2006


Hi, Joaquim,

Now I have exact the same problem you claimed in your email which I copied
at the end. Did you find out the solution for fighting the high abundant
clones? Could you please send me the protocol of yeast colony hybridization
that works well in your hand. Thank you very much for your help.

Best regards,

Wei Zhou
Rheumatology Unit
Karolinska Institute
Stockholm Sweden

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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