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Homologous recombination in yeast

Homologous recombination in yeast - Yeast Forum

Homologous recombination in yeast - Discuss research on yeast the model organism. Post questions and topics on the molecular biology and genetics of Saccharomyces cerevisiae and other yeast species.


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Old 06-28-2010, 02:54 PM
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Default Homologous recombination in yeast



Hi,

I'm going to be starting homologous recombination in yeast soon and have been hearing that it's quite difficult to get it to work. If anyone has any hints and tips, any awesome protocols, or any trouble they had and how they fixed it, it would be very much appreciated. I've never done it before so any advice would be welcome.

Thanks,

Cathy.
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Old 06-28-2010, 09:41 PM
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Default Re: Homologous recombination in yeast

I have a sure-fire method...just did a few KO with it this weekend:

Grow 5 mL of culture to exponential (I am usually around .4 to .6).

Spin down, wash with water, spind down, then resuspend in 100 uL of 1X LiOAc (100mM I believe)

Pipette off the LiOAc and then resuspend pellet in:

240uL 50% PEG 3350
25uL ss DNA
45uL PCR product
36uL 10X LiOAc

Incubate at 30c for 45 minutes, then move to 45c for 25 minutes. Wash with water and plate all onto YPD. Replica-plate onto selection the day after.

I am usually not using auxotrophic markers, but if that is what you are using you can skip the YPD and replica-plating.

This method is for PCR product also, I usually run a 50uL PCR and then take about 4uL for a gel to check, then just put all of it into the transformation.
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Old 07-02-2010, 12:50 AM
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Default Re: Homologous recombination in yeast

I meant 42c not 45c of course for that last incubation...
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