Sex on a plate is as easy as it sounds:
Assuming your mutations don't severely affect mating frequency, you can
simply take a colony of each strain with a toothpick, and mix the two
together on a rich media (nonselective) plate -- just mush them together
and spread them around to make a small "patch" on the plate. Now put this
plate at 30 degrees overnight, and then next day replicaplate (using
sterile velvet, etc.) to a plate lacking met and trp. Alternatively, you
could scrape up all the mixed cells and resuspend them in water and plate
them with a spreader. In the former case, be sure to take the colonies
that are coming up within the "patch" and re-streak them for single
colonies on a -met -trp plate.
Of course, some good controls wouldn't hurt -- mixing the two haploids
with themselves instead of with each other, and making sure that the Met+
Trp+ colonies that come up are in fact diploid (don't themselves mate,
larger cells, bipolar budding, etc.). And, to be positively certain, you
could do Southern or PCR analysis on the resulting strain to check for
both knocked-out alleles.
This works great for everyone in our lab, so I think it should work for
you. There are more "quantitative" mating assays that are also more
complicated; any paper that describes such a quantitative mating assay
should have the protocol or a reference thereto.
On 18 Jan 2000 darren.wells at bbsrc.ac.uk wrote:
> Dear All,
> I am wanting to combine two Saccharomyces cerevisiae strains of opposite
> mating type. The first has a gene inserted into its genome which I am
> intressted in. The second in a knockout of a different gene which my
> supply a good background to test the activity of the inserted gene.
> Does anyone have a protocol for bringing these two together. My
> knowledge and experience of yeast is limited so something simple would
> be a great help.
> The two strains are;
> his 3delta1; leu 2delta0; met15delta0; ura 3delta0
> leu 2-3; leu2-112; deltatrp1; ura3-52
>> I think that a diploid may be selected via the trp and met autotrophies
> Thanks for your help.
>>nick.cryer at bbsrc.ac.uk>> ---
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