Yeast Labs 2023-F

Yeast Labs 2023-F kdorfman Thu, 07/20/2023 - 18:07

Here are all the prep pages for the yeast genetics labs 2023

List of yeast strains

Yeast media

A Classroom Guide to Yeast Experiments

Available for purchase from Carolina. (We have a copy available in a 3-ring binder.)

Week 01 - no labs

Week 01 - no labs kdorfman Wed, 08/09/2023 - 19:38

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)

Week 02 - Yeast mating

Week 02 - Yeast mating kdorfman Thu, 07/20/2023 - 18:16

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):

station activity materials
1 label plate YED plates, labeling template, Fisher Finest marker
2 plate HA1 across 3 small plates of HA1 on YEPAD, sterile toothpicks, used toothpick bucket
3 plate HA2 across 3 small plates of HA2 on YEPAD, sterile toothpicks, used toothpick bucket
4 plate HAR across 3 small plates of HAR on YEPAD, sterile toothpicks, used toothpick bucket
5 Plate HB1 down, mixing carefully with the A strain already there, new toothpick each time 3 small plates of HB1 on YEPAD, sterile toothpicks, used toothpick bucket
6 Plate HB2 down 3 small plates of HB2 on YEPAD, sterile toothpicks, used toothpick bucket
7 Plate HBR down 3 small plates of HBR on YEPAD, sterile toothpicks, used toothpick bucket

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

A types alpha (B) types
HA0 (WT) HB0 (WT)
HA1 (ade1) HB1 (ade1)
HA2 (ade2) HB2 (ade2)

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.

Week 03

Week 03 kdorfman Mon, 07/24/2023 - 20:49

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

Week 04

Week 04 kdorfman Mon, 07/24/2023 - 20:50

Yeast Labs week of 9/25/23

(See also seedling observations)

Pipetting exercises:

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??

Week 05

Week 05 kdorfman Mon, 07/24/2023 - 20:50

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

Week 06 - no labs

Week 06 - no labs kdorfman Wed, 08/09/2023 - 19:39

No Labs week of 10/9/23

Indigenous peoples day

Make up labs if needed

Week 07

Week 07 kdorfman Mon, 07/24/2023 - 20:50

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

Week 08

Week 08 kdorfman Wed, 08/09/2023 - 19:37

Yeast labs, week of 10/23/23

Yeast genetics sequencing recap;

(See also F2 observations and dog DNA extraction))

Yeast genetics practical exam

Yeast ADE primers & pcr

Yeast ADE primers & pcr kdorfman Mon, 08/21/2023 - 14:06

Yeast Primers (Carolina stocks)

Gene primers 1 product size (bp)
Ade 1 5’ – GTTGGGTTTTATCTTTTGCAGTTGG - Ade1f JW1b
5’ – GGCGACTTGTAGTATATGTAAATCACG -Ade1r 1040
Ade 2 2 5’ - ATTCTCCTGCCAAACAAATAAGCAACTC - Ade2 F25
5’ – ACATTCCGCCATACTGGAGGCAATAA - Ade2 R26 1010
Ade 2 5’ - TTGCCAATGCCAAAGAATTTCACATC - Ade2 F29
5’ - GCATTGAGCCGCCTTATATGAACTGT - Ade2 R30 1085

Master Mix

Reagent uL
2x buffer (4) 20
water 12.8
NEB Taq 0.2

Reaction Mix

Reagent uL
master mix 33
F primer 2
R primer 2
DNA uL to get 50 ng
(water uL to get to 40 uL)

Buffer 4 3 1X, pH 8.4

reagent concentration
Tris-HCl 14 mM
KCl 70 mM (standard taz buffer is 50 mM KCl)
MgCl2 3 mM
dNTP 65 uM each

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

step temp time
1 94 30 s
2 93 15 s
3 61 20 s (-1C/cycle)
4 go to #2 4 times
5 93 15 s
6 57 20 s
7 72 12 s
8 go to #6 29 times
9 72 5 min
10 10 hold

  1. the forward primers of each gene just miss the ATG start because of the lack of G & C bases outside the cds. ↩︎

  2. the 1.7 k.b. Ade2 cds was broken up into two PCR reactions overlapping by 300 bases because of its size and uncertainty over the average size of strands in our crude yeast gDNA prep, but the PCRs seemed to work well with 50 ng, and it might be possible to do the Ade2 in one PCR. ↩︎

  3. This is exactly the same as 1x standard taq buffer, but the [KCl] is slightly elevated to help the GC-poor primers anneal. I have just added KCl to the regular reaction before with no problem. (John Willoughby) ↩︎

Yeast primers Dharmacon

Yeast primers Dharmacon kdorfman Thu, 09/07/2023 - 17:01

Recommended primers for Dharmacon yeast stocks

gene notation up down
ADE1 Dhar HA1 AGAGATCAGCCAGACTCGTC
ADE2 Dhar HB2 CCTTCATTGACACTGATCTG GAGACCAGAACACTTGCCTA