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[dcadieux]

I am just starting my first attempts at yeast transformation in preparation for a Y2H screen.  After two attempts withe Clontech Yeast Transformation protocol, I have yet to have any colonies appear.  After talking with their technical support, which wasn't that helpful, I found out that the DMSO I was using is not molecular biology grade.  What is the role of DMSO in the transformation protocol and could this be responsible for my zero colonies?

[perneseblue]

dcadieux, on 26 July 2010 - 09:24 AM, said:

I am just starting my first attempts at yeast transformation in preparation for a Y2H screen.  After two attempts withe Clontech Yeast Transformation protocol, I have yet to have any colonies appear.  After talking with their technical support, which wasn't that helpful, I found out that the DMSO I was using is not molecular biology grade.  What is the role of DMSO in the transformation protocol and could this be responsible for my zero colonies?

It is believed that that DMSO's role is to permeabalise the yeast cell wall. A more permeable cell wall, would allow improve uptake of DNA.

It is certainly possible, but unlikely that the quality of DMSO is at fault here and now. Today, the chemicals  you can buy from a company like Sigma or Fisher scientific are usually pure enough to get your experiment to work... (not at maximum efficiency perhaps). What grade of DMSO are you using?

Please, note yeast cells after passing through the transformation protocol are fragile and will need a few hours to recover. Thus these cells can  burst from sudden changes in osmotic pressure.