Yeast Competent Cells - (Feb/06/2008 )

I am attempting to set up a yeast one hybrid screen and am doing some control transformations to get a feel for the techniques. The problem is I am only getting transforming efficiencies of 3.2*10^3 sing a pGBT9 control plasmid and Y187 strain. I am using a LiAc method detailed in the product manual I am using
(http://www.clontech.com/images/pt/PT3529-1.pdf).

Any tips on preparing the competent cells/how to handle them or how not to !/spreading them/preparation of media (some manuals/reports are telling me to autoclave stuff other sources are telling me I cant !) would be greatly appreciated.

thanks in advance

I also used the Y187 strain and I didn't manage to get the TRAFO protocol to work that well (it probably needs to be optimized for each strain), but my default protocol worked fine.

About autoclaving, I believe I autoclave everything... I just autoclave the minimal medium for selection (CM in my case) at 110ºC and pressure slightly inferior to 1bar.

Do make sure your PEG stock remains closed and prefferably sealed - the transformation is very sensitive to this.

Are you using 1ug of library for each transformation? I've had some problems with quantifying libraries for this (with spectrofotometer). Say I would load 1ug (according to the spec) of two different libraries in a gel and the intensity would be really different. I was just lucky I had another library in the lab so I calibrated the other ones with it through gel intensity. After that the transformations started working much better. You can try doing some transformation tests with different volumes of library to see what's the minimal amount that gives you a good efficiency.

I say always denature your carrier DNA even if it says "denatured" on the tube - it's only 10min so it won't be that much trouble.

That's all I can remember for now, lets hope any of this can help you...

Thanks for the input.I have had another go at transforming a control plasmid and am still not getting the efficiencies I should (approx an order of magnitude too low). Are there any specific tips for handling yeast cells i.e. when to vortex cells vs when not to ? is pipetting cells ok ? should I treat them as I would a bacterial competent cell ?

cheers for the help

I have not done a systematic study on the yeast transformation protocol. But I have found that it is important to harvest the cells at around OD 0.5. By OD 0.7-0.8 transformation efficiency becomes unacceptable.

I have also found that transformation efficiency is improved by being fast.

I don't vortex my yeast cells, instead I tap my cells to mix. I also I keep the centrifuge speed as low as possible to avoid rupturing the cells. If you see the appearance of a black dusting it is probably mitochondria from ruptured cells, a hint to reduce the centrifuge speed. Finally I also keep my cells on ice - I don't know if keep the cells cool actually helps though.

I have the feeling that the recovery stage is actually quite important to get more transformed colonies. Yeast do not like overly 'dry' plates, and seem to recover and grow much better in humid incubators. Soft agar has been used to improve recovery rates... but I do not have any experience with this variable. You could give soft agar a try.

There is a better transformation protocol using lyticase. It allow transformation of very large DNA fragments in to yeast, but the protocol is long winded.

Sorry to hear that you're still having troubles. When you make your competent cells, do you take OD readings, or do you count your cells under a microscope? Counting is much more accurate than OD readings because yeast cells grow at different rates and densities and clumping can give you inaccurate measurements. When I do my transformations, I inoculate my culture at 2 x 10^6 cells/ml, then grow them until the culture becomes 5-6 x 10^6 cells/ml. This can take anywhere between 2-4 hours depending on the strain. Also you might want to check the quality of your DNA.

Thanks for the tips, I am just measuring my cells using OD but I am vortexing before doing so to try and reduce the clumping. I havent checked my DNA quality but it coming from a company so I dont expect any problems but I will do it anyway. Trying another transformation this week so fingers crossed !

You may want to clean the pGBT9 control plasmid even if it did come in the kit. I am also learning how to do yeast one hybrids and have had troubles with the transformation efficiency of the control prey plasmid pGAD-Rec2-53, but the efficiencies are fine with the other plasmids (pGBT9, pHIS2.1 and p53HIS2). I remade the -Leu media and did the control experiments again with no improvement so I called Clontech and they suggested reprecipitating the plasmid. I am testing the cleaned pGAD-Rec2-53 this week...

Good luck!

Hi kzin,

Thanks for the tips ill have a look at the plasmid on a gel and our nanodrop to see whats what. If youve got any other tips e.g handling the cells,mixing or making media (im reading about lots of different methods especially about what to and what not to autoclave) or about anything really I would be most grateful as im close to pulling my hair out.

Best of luck with your transformation

steve

You can also prepare electrocompetent yeast cells. I can post the protocol if you are interested.