Diploid Generation and Sporulation

For Diploid Generation:

1.         Obtain strains 210 and 216 (his+leu-ura-ade-) from Rory’s yeast strain box (-80 freezer) and mix on emm complete plates with drop of sterile water.  Culture plates at room temperature.  24 hrs yields the whitest colonies in my hands.

2.         Take samples at 6 / 12 / 24 hrs spread thinly on emm + leu / ura (- ade).  Culture plates at 37 C for several days.  Diploid colonies will appear white (while non-diploid colonies will grow slowly and appear red).  These colonies may be undergoing sporulation.  Don’t worry and continue with the protocol.

3.         Pick white colonies and spread on YES + 5 mg/L of Phloxin B (stock 5g/L).  Diploid colonies will be red.  Diploid colonies should be stable on YES, yet do not keep well at 4 C, therefore make fresh for experiment.

4.         At this point, diploids are ready for transformation. 

5.         For gene disruption, proceed normally as if the cells were haploid.  Take care to select for white colonies on Emm (-Ade) to ensure your cells remain diploid throughout the transformation procedure.

Sporulation:

1.         Once you have successfully transformed your diploids, you are ready to sporulate your cells.  This is very easy.  Simply spread a lump of cells on Emm complete plates and culture for 2-3 days at room temperature.  Monitor their progress under a microscope during this period.  70-80 % of the cells should sporulate successfully under these conditions.

2.         You are now ready for tetrad analysis.  For this, you will need a dissecting scope modified for tetrad dissection and someone skilled in this technique to give you a hands-on lesson.

Any questions, please contact:

Rory Geyer

617-432-2065

rgeyer@hsph.harvard.edu