In order to mate efficiently, the two strains must be of opposite mating
type, MATa or MATalpha. Then you can mix a bit of each strain and select
for diploids, such as by trp and met auXotrophy. If they are the same
mating type, then you'll need to change the type of one of the strains,
such as by introducing the homothallate (HO) gene product. Look in your
local library for Methods in Genetics, volume 194, edited by Guthrie and
Fink. This has chapters on mating and selecting diploids. You may need to
eventually sporulate the diploid and select for a haploid that has both
of your genes (or mutation) of interest, as the diploid will be
heterozygous for both. Note that by the time you do all this, you may
have had an easier time deleting the second gene in the first strain.
This is easy nowadays with DNA oligomers, PCR and the correct plasmids.
See Longtine et al., (1998) Yeast 14, p.953.
Finally, if you have not had experience with yeast, I'd suggest to comb
your local university for a yeast genetics and molecular biology lab, and
take the professor out for a beer to talk about your experimental design
before you start. Preparation time now will save you tenfold later.
In article <8620u9$rav$1 at mercury.hgmp.mrc.ac.uk>,
<darren.wells at bbsrc.ac.uk> wrote:
>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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