Yeast deletion haploid test protocol

The strain of interest should be streaked or spotted onto the appropriate
minimal media agar with G418.  Mat a plates were spotted onto minimal media agar
with G418 (200mg/liter) minus methionine.  Mat alpha plates were spotted onto
minimal media agar minus lysine.  Each was spotted with a mat a control, a mat
alpha control, and a diploid control.


Minimal media recipes
10x Amino Acid Mix 
(Makes 1 Liter)
0.2 g		Adenine Hemisulfate
0.2 g 	Uracil
0.2 g		L- Tyrosine
1 g 		L- Tryptophan
1 g 		L- Histidine - HCl
1 g		L- Arginine - HCl
1 g		L- Leucine
1 g		L- Isoleucine
1 g		L- Phenylalanine
1 g 		L- Glutamic Acid
1 g		L- Aspartic Acid
3 g		L- Valine
4 g		L- Threonine
8 g		L- Serine
976 ml	dH2O

To 500 ml of dH2O add all of the dry/powder constituents (amino acids).
Add 476 ml of dH2O.
Mix/Shake well - may require heating on a stir plate.
Do not autoclave, filter sterilize the 10x Solution using 0.2 micron filtration
units.  


10x (20%) Glucose Solution (100 ml)
20 g		D-(+)- Glucose/Dextrose
80 mL		dH2O

Mix and stir well on a stir plate.
Filter sterilize the 10x Solution using a 0.2 micron filtration unit.



10x Ammonium Sulfate (100ml)
5 g 		Ammonium Sulfate
95 mL		dH2O

Mix and stir well on a stir plate.
Filter sterilize the 10x Solution using a 0.2 micron filtration unit.

10x Yeast Nitrogen Base (100 ml)
1.7 g  	Bacto Yeast-Nitrogen Base (w/o AA or AS)
98.3 mL	dH2O

Mix and stir well on a stir plate.
Filter sterilize the 10x Solution using a  0.2 micron filtration unit.
 

Agar stock (600 ml)
20 g		Bacto-Agar
580 mL	dH2O

Mix.  Autoclave for 20 minutes at 121C

Single Amino Acid Solutions (100 ml)
1 g		Lysine OR Methionine
99 mL		dH2O 

Mix and stir well on a stir plate.
Filter sterilize the 10x Solution using a 0.2 micron filtration unit.


Yeast CM Agar (1 liter)

Melt 600 ml of Agar stock in the autoclave - 105C for 15 minutes and warm at
80C.  Do not let the agar cool - use at 80C.
Aseptically add the following to the agar (in order-swirl after each component):

100 mL of 10x Ammonium Sulfate
100 mL of 10x Glucose Solution 
100 mL of 10x Yeast Nitrogen Base
100 mL of 10x Amino Acid Mix
3.3 mL of Geneticin (G418) antibiotic (300x stock is 60mg/ml)(working=200ug/ml)
Add EITHER 2 mL of Methionine solution (for testing a haploids) or 3 mL of
Lysine solution (for testing alpha haploids)

Mix well and pour plates.  Allow the plates dry completely at room temperature
under the hood for 24 hours then on the bench top - 2 to 3 days is recommended.