Yeast Labs 2023-F
Yeast Labs 2023-F kdorfman Thu, 07/20/2023 - 18:07Here are all the prep pages for the yeast genetics labs 2023
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 - 19:38No 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 - 18:16Yeast 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.)
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 - 20:49Yeast 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 - 20:50Yeast 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??
Week 05
Week 05 kdorfman Mon, 07/24/2023 - 20:50Yeast 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
ADE sequences
ADE sequences kdorfman Fri, 08/25/2023 - 16:39Week 06 - no labs
Week 06 - no labs kdorfman Wed, 08/09/2023 - 19:39No Labs week of 10/9/23
Indigenous peoples day
Make up labs if needed
Week 07
Week 07 kdorfman Mon, 07/24/2023 - 20:50Yeast 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 - 19:37Yeast 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 - 14:06Yeast 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 |
-
the forward primers of each gene just miss the ATG start because of the lack of G & C bases outside the cds. ↩︎
-
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. ↩︎
-
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 - 17:01Recommended primers for Dharmacon yeast stocks
gene | notation | up | down |
---|---|---|---|
ADE1 | Dhar HA1 | AGAGATCAGCCAGACTCGTC | |
ADE2 | Dhar HB2 | CCTTCATTGACACTGATCTG | GAGACCAGAACACTTGCCTA |