Yeast Mating test

 

Strain: RFY206/SH(lacZ plasmid): Mata. Ura3, his3, trp1, leu2

             EGY48:  Mata. Ura3, his3, trp1, leu2

 

 

1.      Introduce Trp1 (B42) plasmid into yeast strain EGY48 and select transformants on

Trp- glu plates.

2.      Introduce His3 (Lex) plasmid into yeast strain RFY206 containing SH plasmid and select transformants on Ura-, His-, glu plates. : The RFY strain grow slower than the EGY strain

 

Method 1

3.      Inoculate 3-4 colonies from each transformation event into a sterile effendorf tube containing YPD broth. (See note 1.) And vortex.

4.      Dispense 10ul of YPD containing yeast cells into wells of a 96 well plate so that each well contains one B42- and one lex harboring cells. Mix them well by inoculating tip or toothpick.

5.      Grow them at 30oC for a full day.  To increase possibility of contact of mating cells, keeping YPD vol at minimum is important.

6.      Spot 3-5ul of broth from each well to master selection plates (ura-, his- trp- Glucose) and grow them for 2 days.  You may see several separate colonies or cell lawn depending on how well mating did.

7.      Transfer each colony from the master plate to a set of selection plates.

A.      Ura-, his-, trp-/Gal, X-gal

B.       Ura-, his-, trp-/Glu,Raff X-gal

C.       Ura-, his-, trp-, Leu/Gal, Raff

D.      Ura-, his-, trp- Leu-/ Glu

8.      Grow them at 30oc for several days and check the color and growth pattern.

 

Note 1. If you have 6 B42 and 10 LexA plasmids to test, suspend each B42 plasmids in (10+1)x10 ul of YPD and each LexA in (6+1)x10ul.  Then make an aliquot of each 10ul of B42 in one law and each 10ul of Lex in one column on 96 well plate.  When you finish all dispense each well contains mixture of two different Lex, and B42 plasmids.

 

Method 2

3. If transformed plate was stored at 4oC for more than a week, inoculate several colonies from one transformation event into a proper minimal medium and incubate them at 30oC.

4. After a day, mix each of EGY and RFY strains (about 20-30 ul) in effendorf tubes, centrifuge, remove medium and suspend the with 50 ul of YPD and incubate for overnight.

5. Centrifuge tubes to remove YPD and resuspend cells with sterile water and plate onto U-H-T-/Glucose plate. (You can transfer each content in the well of 96 plates and replica directly to the selection plates of step 7. Go to step 8 or 7 depending on what you did.