Yeast

Strain Thermo Scientific (now GE Dharmacon)

YSC6273-201920294

Mata YIL015W

http://dharmacon.gelifesciences.com/search/?term=YSC6273-201920294

Store in -80

Growth Protocols http://www.sigmaaldrich.com/technical-documents/protocols/biology/yeast…

Streak on YPD plate. Allow 48 hours at 30C.

Hemocytometers

InCyto C-Chip, DHC-N01-5 Neubauer improved

Fisher 22-600-100 50 for $90.78

Growth medium:

http://mcb.berkeley.edu/labs/koshland/Protocols/Media/yepdmedia1L.html

http://wahoo.cns.umass.edu/content/ypd

Per L:

• in 900 mL H20. Autoclave

  • 10 g yeast extract
  • 20 g peptone

• In 100 mL H2O. Filter sterilize

  • 20 g glucose

Mix

Plates

http://mcb.berkeley.edu/labs/koshland/Protocols/Media/yepdplates.html

Per 100 mL:

• 1 g yeast extract
• 2 g peptone
• 2 g agar
• 90 mL water

autoclave.

• 10 mL 20% sterile filtered glucose

Ingredients:

http://mcb.berkeley.edu/labs/koshland/Protocols/YEAST/Arrestingyeast.ht…

http://www.zymoresearch.com/yeast/specialty-products/alpha-factor-matin…

http://www.sigmaaldrich.com/catalog/product/sigma/t6901?lang=en&region=…

http://www.sigmaaldrich.com/catalog/product/sigma/h8627?lang=en&region=…

Concentrations

ingredient	working conc	MW	stock
nocodazole	0.015 mg/mL	301	3 µL 33 mM (= 10 mg/mL)
HU	0.2 M	76	Koshand lab 2M in H2O
alpha factor for BAR yeast	3 µM	1684	Koshand lab 1mM in 1N HCl
alpha factor for bar- yeast	15 nM	"	"

Calculations

• Nocodazole:

To make 50 mL YPD with 0.015 mg/mL nocodazole
25 (!) 3 µL aliquots 10 mg/mL noc
vi = (0.015 mg/mL * 50 mL)/10 mg/mL = 0.075 mL

1.5 µL 10 mg/mL stock per mL medium

• Hydroxyurea: (Sigma H8267-1g)

To make 50 mL YPD with 0.2M HU
vi = (0.2M * 50 mL)/2M = 5 mL

100µL 2M stock per mL medium

Buy 1 g, make 6.6 mL 2M stock
MW = 76, so 1 g = 1/76 mol = 0.0132 mol
vol = (1000 mL/2 mol) * 0.0132 mol = 6.6 mL

• alpha factor

To make 50 mL alpha factor at 3 µM from 1 mM stock
vi = (3 µM * 50 mL)/1000 mL = 0.15 mL

Can buy 0.5 mg
To make 1mM stock from 0.5 mg
0.5 mg* (1 mmol/1684 mg) = 0.000297 mmol
vol = (1000 mL / 1 mmol) * 0.000297 mmol = 0.297 mL 0.1N HCl

3 µL 1mM stock per mL of medium for 3 uM

0.015 uL 1mM stock per mL medium for 15 nM

Arrest protocol:

http://mcb.berkeley.edu/labs/koshland/Protocols/YEAST/Arrestingyeast.ht…

Allow 3 hours in drug to arrest.

morning before class

(An overnight culture may not be dense enough!!)

Start ~5 mL ON culture from plate. shake, no heat

9 am, day of class

10 am

• Aliquot 5 mL YPD into each of 4 tubes.
• Control. Do nothing
• Nocodazole. Add 7.5µL nocodazole stock
• HU. Add 500 µL stock
• alpha factor. Add 15 µL stock
• add enough cells to get slightly cloudy
  • OD at 1 pm should be ~0.2
  • So at 9 am, should be ~0.1
• incubate at RT in tube roller (from biochemistry)

Just before class

• Check for concentration and clumpiness

  • if necessary dilute
  • sonicate (borrow sonicator from biochem) if necessary: 20% 30 sec 12:30 pm, just before class

• Make 6 ~0.7 mL aliquots each culture into microfuge tubes

• One set of 4 per table.
• Each pair does all 4; table shares the samples.