No labs on weeks with a holiday

If necessary, amplify the yeast strains students will need for Yeast 1 on YEPAD. Grow up from frozen if necessary.

• HA0 (demo only)
• HA1
• HA2
• HAR
• HB0 (demo only)
• HB1
• HB2

Plate all strains on YED demo plates (all 4 A on one plate, all 4 B on the other), 1 each per table.

Plate strains students will mate (1,2,R) on YEPAD plates, 1 each per pair, (all 3 A on one plate, all 3 B on the other)

Yeast labs, week of 9/11/23 (2nd week of classes, 1st complete week)

See notes here

Total YED plates for the week for 4 sections plus prep: 75

Students mate 3 A mating types with 3 alpha mating types

Take YED plates out early so they stop sweating.

Make an assembly line of 7 stations (use templates):

For mating, students need these strains on YEPAD plates so they all start out white:

• HA1
• HB1
• HA2
• HB2
• HAR
• HBR

For observation, students need these strains on YED plates so their colors can be observed:

• HA0
• HB0
• HA1
• HB1
• HA2
• HB2
• HAR
• HBR

For observation of colors, make demo plates (maybe 4 of each):

• 4 YED plate with 4 A strains
• 4 YED plate with 4 alpha (B) strains

(If you let the parental strains sit on YED more than a couple of days, the mutant strains turn red, and the red color will still be there even after there are heterozygous diploids with complementation.)

List of yeast strains

Start 6 strains at least the Friday before the first lab.

Instructors should make some mating plates to have in the fridge to hand out when student plates are bad.

Yeast Labs, week of 9/18/23

(See also planting seeds)

• Examine mating mixtures

• Take picture for notebook

• Record colors in notebook

• Replicate YED mating plate onto 1 MV plate per pair

  • sterile velvets
  • replicating plate stampers
  • elastic hair ties
  • bleach bucket for used velvets

• Sample mating yeast for schmoos and diploids; take picture for notebook. Set up mating mixtures in liquid YEPAD 2 hours before class.

48 MV plates

Yeast Labs week of 9/25/23

(See also seedling observations)

Pipetting exercises:

• Colorimetric pipetting exercise
• Weighing water
  • 1 balance per pair
  • weigh boat
  • beaker of dH2O
  • extra batteries

DNA extraction from yeast

• Reagents
  • 200 mM LiOAc, 1% SDS (4x 100 uL/pair)
  • 95% EtOH (4 X 300 uL/pair)
  • 70% EtOH ( 4 X 400 uL/pair)
  • TE ( 4 x 80 uL/pair)
• Equipment
  • microfuge tubes
  • hot block at 70C
  • centrifuges for 1.5 mL tubes, 15,000 x g
  • paper towels
  • Nanodrop

Sequences

• send out for sequencing?
• 4 strains per table
• really? or pull sequences out of her back pocket?

Check MV replica plate from last week

• check diploids
• draw picture
• label
• transfer
  • some??? to YED (to check the next day, next day toothpick to YEKAc plate 1/pair (=48)
  • pick one to YED (class "pre-spore")

YED ?2 per pair? = 48

YEKAc??

Yeast Lab week of 10/2/23

(See also pollination)

YED plates: 4/pair

Mutagenesis

See notes here

• Sterile technique
• Spray 70% ethanol
• Serial dilution (5 tubes)
• Sterile water
• glass beads
• Wild type stock plate (HA0 or HB0) (1/table)
• YED plates 3/pair
  • control (most dilute)
  • 7 sec UV exposure (next most dilute)
  • 10 sec UV exposure (next most dilute)
• UV transilluminators
• face shields

Come in next day and move plates to fridge

Look for spores

• check YeKAc plate from previous lab; make a wet mount and look for spores
• Microscopes
• slides
• coverslips
• water
• pick from YeKAc plate to YED plate (1/pair) to observe for color next lab

Observe class "pre-spore" YED plate from previous week

No Labs week of 10/9/23

Indigenous peoples day

Make up labs if needed

Yeast Labs week of 10/16/23

See also Observing F2

Count colonies on UV irradiated plates

Check color on YED plate from YeKAc plate

Yeast labs, week of 10/23/23

Yeast genetics sequencing recap;

(See also F2 observations and dog DNA extraction))

Yeast genetics practical exam

Yeast Primers (Carolina stocks)

Master Mix

Reaction Mix

Buffer 4 ³ 1X, pH 8.4

Program Y-TD1 (anneal 61C -> 57C then 57C)