yeast transformation failed after doing it many times - (Nov/11/2007 )

Hi,

I am trying to optimize my yeast two-hybrid protocol for cDNA library screen. I was able to achieve a transformation efficiency of .25 x 10^6 cells/ ug DNA awhile ago. But, recently I haven't been able to achieve a number even close to that. Have anyone experienced that and/or has any suggestion on what I should do? Thanks.

When I did my Y2H screen i used the TRAFO transformation protocol:

http://home.cc.umanitoba.ca/~gietz/2HS.html

One thing you may consider is that when you scale up the reaction the cfu/ug can drop dramatically so it can be better to do several (or many) small transformations with 1-2 ug of DNA than, say, one large transformation with 20ug.

Also, are you creating your strains by co-transformation or mating? If co-transformation, putting in a second plasmid can drop the efficiency of the protocol if there is already a plasmid in the strain.

Once I had the same problem (though with yeast one-hybrid) and I solved it by making a new PEG 50% stock.
The thing is that yeast are very sensitive to the concentration of this compound, and if water evaporates and its concentration changes, the transformation efficiency can be severely diminished.

Other than that I'd say make sure the carrier DNA is really denatured and confirm the cDNA library concentration not only by spec, but by running it on a gel. I've had libraries with a big Abs260 and then, upon running on a gel, I'd realize that the concentration was far lower...

Hope this helps...

Hi,

Thanks for the help, I made new PEG 50% stock and tried transformation again. And my transformation efficiency improved but not to 0.25 x 10^6 cells/ug DNA. I guess I have to keep doing it.

Hi,

I have a question about the TRAFO protocol. When you used this protocol, did you change any of the steps in order to reach a high transformation efficiency? I tried TRAFO before but it hasn't been helpful in generating many transformants for me. If you have any suggestions, I would really appreciate the help. Thanks a lot.

I once used TRAFO too, but it didn't work all that well for my yeast strain. I think it has to be optimized according to the strain you're using. If you have a good and reliable protocol for your strain I'd say to stick to it.