2023-Fall kdorfman Mon, 07/17/2023 - 19:45

John Willoughby & Jedi Chilufya

Week date Yeast Fast Plants Dog
1 9/4 NO LABS
2 9/11 Yeast 1 observe & mate
3 9/18 Yeast 2 replica plate plant genetics 1; plant seeds
4 9/25 micropipetting;
Yeast 3
DNA extraction, txfer to YED, then to YEKAc
F1 observations
5 10/2 Yeast 4 (contig assembly ADE1, ADE2 sequences); UV mutagenesis, pollination
6 10/9 NO LABS collect dog DNA
7 10/16 Yeast 5 hunt for mutants, Yeast practical exam observe F2
8 10/23 observe F2 Dog DNA extraction
9 10/30 collect & plant F2 seeds Dog DNA quantification, PCR, Molecular basis of dog coat color
10 11/6 observe F2 who's the father? Prep MC1R sample for sequencing, gel electrophoresis
11 11/13 work on reports dog TRYP1 locus, analysis of MC1R genotype & sequence, cap3 sequence assembly
13 11/27 bioinformatics
14 12/4 Dog genotype/phenotype presentations
(15) 12/11 finals week report due

Google Folder for course

Dog Genetics Labs 2023-F

Dog Genetics Labs 2023-F kdorfman Thu, 07/20/2023 - 18:08

Here are the prep pages for the dog genetics labs 2023

Puritan Try Cap-shure Swabs with attached caps from McKesson

Canine Coat Color Basics

Dog coat color genetics chart

coat color basics

lhasa apso coat color

Dog Cell Lines

Dog Cell Lines kdorfman Fri, 09/01/2023 - 15:06
Line # breed defrost frozen? # swabs notes
104 Newfoundland (M) 8/28 Very few adherent cells as of 9/1
104 Newfoundland (M) 8/31 10
4 from spun down culture medium
huge numbers of floating cells (made swabs from liquid)
105 golden 8/28 6 from spun down culture medium very few cells overall, adherents are round
106 Maltese 8/28 8/30 15
3 10/23/23
huge numbers of cells. put into 2 25 cm2 flasks>. tested regular DMEM in 12.5 cm2 flask. 1 25 cm2 flask to freeze
109 terrier mix? 8/28 about 2 adherent cells per field of view at 10x
110 pointer 8/28 single digits adherent cells
IPC 366 1 ? 8/28 9/1 9 grew like gangbusters! 3 swabs per 12.5 cm2 flask. froze one
thawed and grew up for swabs. Buckets of DNA!! (multinucleate). Froze 2 more vials.
112 Yorkshire terrier 11 YO F 9/8/23 6
15 from trypsinized T75 flask
Grow great!
thawed and grew, made swabs, then they petered out. Not able to amplify enough to freeze another vial.
113 Beagle (Thelma) 6YO F 9/12/23 10 from trypsinized T75 flask great growing cell line, but started to fail after split. Maybe seeded too thinly? Split them all to a smaller flask 9/27 to see if they will grow up to large enough numbers to freeze

Link to Kathleen's photo gallery

Frozen cell lines

Dog DNA summary

Dog DNA summary kdorfman Wed, 08/02/2023 - 14:47


letter gene full name traits
E MC1R melanocortin 1 receptor eumelanin (requires signal molecule from agouti or defb) vs. pheomelanin
ee produces a non-functioning MC1R
K defb103 defensin beta 103 (= CBD103 beta-defensin 103) dominant black has a glycine deletion
KB makes a signal that binds tightly to MC1R, preventing agouti expression
B TyRP1 tyrosinase related protein 1 black (B_) vs brown (bb)
A ASIP Agouti Signaling Protein ASIP promotes pheomelanin (aa: no signal, A_ signal with varying on-off patterns)

MC1R Alleles

letter gene full name traits

Agouti Alleles

letter gene full name traits

NOTE: If you have the nucleotide sequence for an individual and the reference amino acid sequence, to compare them:
Query: nucleotide sequence
Subject: reference amino acid sequence

Agouti sequences

Agouti sequences kdorfman Mon, 08/21/2023 - 14:08


To find the crucial 82, 83, 96 amino acids:

_ _ PRPP...CVAT _
82 83 ................. 96

MC1R sequences

MC1R sequences kdorfman Mon, 08/21/2023 - 14:10


TYRP sequences

TYRP sequences kdorfman Mon, 08/21/2023 - 14:09



defB103 kdorfman Mon, 08/21/2023 - 14:10


Week 07 DNA collection

Week 07 DNA collection kdorfman Mon, 07/24/2023 - 21:00

Dog DNA labs week of 10/16/23

Instructions for swabbing dog's mouth

Tell students "Go home and swab your dog"

Week 08 DNA extraction

Week 08 DNA extraction kdorfman Mon, 07/24/2023 - 20:25

Dog DNA labs, week of 10/23/23

Isolate DNA

1 sample per pair, from:

QIAamp DNA minikit 51306


  • Microfuge tubes
  • Sterile forceps (to pull swab from extraction buffer)
  • QIAamp columns
  • collection tubes
  • 56C heat block (10 min)
  • vortex mixers
  • centrifuge 6000 x g 1 min, 20K x g 3 min
  • Nanodrop (may need next class, also) (Need to calculate uL to get 100 ng)

Reagents (per swab)

  • PBS (400 UL)
  • Proteinase K (20 uL)
  • Buffer AL (400 uL)
  • 95% EtOH (400 uL)
  • QIAamp Mini spin column (1)
  • 2 mL collection tube (2?)
  • Buffer AW1 (500 uL)
  • Buffer AW2 (500 uL)
  • Buffer AE (50 uL)

Staff makes spreadsheet on Google Drive with:

  • sample # for each dog
  • concentration
  • quality

DNA from cultured cells

DNA from cultured cells kdorfman Tue, 08/22/2023 - 16:04

From QIAamp DNA Mini & Blood Mini Handbook, Apendix B, p 50

Do not use more than 5 x 10^6 cells

Important points before starting

  • Do not use more than 5 x 106 cells (with a normal set of chromosomes).
  • All centrifugation steps are carried out at room temperature (15–25°C).
  • Use carrier DNA if the sample contains <10,000 genome equivalents (see page 17).

Things to do before starting

  • Heat a water bath or heating block to 56°C.
  • Equilibrate Buffer AE or distilled water to room temperature (15–25°C) for elution.
  • Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared according to the instructions on page 16.
  • If a precipitate has formed in Buffer AL, dissolve by incubating at 56°C.


Week 09 PCR MC1R

Week 09 PCR MC1R kdorfman Mon, 07/24/2023 - 20:25

Dog DNA labs, week of 10/30

students set up 2 identical rxns for MC1R per pair

plus 1 tube each for ASIP, Def103, TyRP1


  • Nanodrop (for students who didn't finish last week)
  • Thermocycler (program: ???)


Instructors make MasterMix + primers; students add 100 ng DNA plus water to make ?? uL

  • Qiagen MasterMix
  • forward primer
  • reverse primer
  • sterile water

Paper activities

  • Complete genetics of dog hair packet
  • Complete MC1R activity
  • Protein/structure/function activity

Week 10 Exo-SAPiT

Week 10 Exo-SAPiT kdorfman Mon, 07/24/2023 - 20:27

Dog DNA labs, week of 11/6

Practice gels to learn well loading

Gel 12 wells MC1R whole class on one gel (100 bp ladder)

ExoSAPIT M1CR 3 tubes/pair

Ready MC1R for sequencing (Ma, Mb, Mc). * 3 tubes per pair (long gene - need to get contigs)

Need Nanodrop again to check concentration

Week 11 Restriction digest

Week 11 Restriction digest kdorfman Mon, 07/24/2023 - 20:27

Dog DNA labs, week of 11/13

Genotyping loci B (TyRP1), E (MC1R), K(defb103)

We provide folder with sequences

Restriction digest TyRP1 ("B" locus) 1 uL/rxn each:

We provide uncut


  • 4peaks to clean sequences Ma, Mb, Mc
  • CAP3 to assemble contigs to get MC1R sequence
  • Clustal also!
  • BLAST to compare to known nt sequences: MC1R (E), Defb103 (K)
  • Expasy (nucleotide to amino acid sequence)
  • Clustal omega to align aa to known sequences

Week 12 - no labs

Week 12 - no labs kdorfman Wed, 08/09/2023 - 19:56

Thanksgiving week of 11/20/23

Week 13

Week 13 kdorfman Mon, 07/24/2023 - 20:27

Dog DNA labs, week of 11/27

Analyze data for A (agouti) locus

  • Expasy to translate sequence to aa
  • CLUSTAL to align aa sequence
    • what aa sequence do you have
    • is it heterozygous?
  • 4Peaks, look for and explain discrepancies
  • Make final hair color predictions

Week 14

Week 14 kdorfman Mon, 07/24/2023 - 20:29

Dog genotype & phenotype presentations, week of 12/4/23 (last week of classes)

Individual completion of dog Practical

Fast Plants Labs 2023-F

Fast Plants Labs 2023-F kdorfman Thu, 07/20/2023 - 18:08

Here are the prep pages for the Fast Plants genetics labs 2023

Fast Plant Resources

Fast Plant Resources kdorfman Thu, 07/20/2023 - 22:00

Fast Plants Life cycle

Fast Plants Life cycle kdorfman Thu, 07/20/2023 - 19:14

Life Cycle diagram

Interactive life cycle illustration

Day developmental event(s)
1&2 germination; seed swells with water until its coat cracks
3 hypocotyl pushes through soil, pulling cotyledons with it; seed coat falls off
4 hypototyl elongates above soil; roots grow down and anchor plant
5-8 True leaves develop; root hairs grow
9-13 Flower buds
14-17 Flowers open; pollination is possible
18-20 Fertilized eggs inside pistils grow to become embryos; pistil swells to become pod
12-40 Petals wilt and fall off; pods dry out. (Stop watering to encourage pod maturation.) When they are completely dry, seeds can be harvested

Fast Plant overall crossing scheme

Fast Plant overall crossing scheme kdorfman Wed, 08/02/2023 - 16:00
allele trait
ANL purple stem
anl green stem (anthocyanin-less)
YGR green leaves
ygr yellow-green leaves
Generation genotype X genotype notes
P anl/anl (anthocyaninless) X ygr/ygr (yellow-green leaf) we plant demos
F1 ANL/anl YGR/ygr X ANL/anl YGR/ygr purchased from Carolina
F2 9 ANL/__ YGR/__:
3 ANL/___ /ygrygy :
3 anl/anl YGR/___ :
1 anl/anl ygr/ygr
grown up from our F1 x F1 pollination

Week 03 Planting Fast Plants

Week 03 Planting Fast Plants kdorfman Thu, 07/20/2023 - 18:14

Fast Plants Day 1 (week of 9/18/23):

(See also Yeast genetics 2)

Students plant seeds of F1 Non-Purple Stem, Yellow-Green Leaf (anl/ANL, ygr/YGR)

These are the offspring of two P1 strains:
Non-Purple Stem, Hairless (anl/anl) X Yellow-Green Leaf (ygr,ygr)

We plant at least 6 pots of non-purple stem parents (P1: anl/anl) for a demo, and ask students to puzzle out the genetics of the other parent "Who's the parent?" One pot per table.

  • 1 pot per student
  • place an inner "mesh" tray into the sink
  • place bucket of soil and an empty tray with mesh insert on cart next to sink
  • place a filled bucket of water by the sink

  • per pair:

    • 15 mL tube of fertilizer pellets (fill tube to ~2ml)
    • microfuge tube of F1 seeds (up to 10 seeds/pair)
    • little paint brush
    • weigh boat
    • labeling tape and marker

According to Carolina,
"F1 generation seed will express dominant traits (purple stems, dark green leaves, standard height) and can be used to produce F2 seed that will show the typical monohybrid (3:1) or dihybrid (9:3:3:1) phenotypic ratios. F2 seed will express the appropriate phenotypic ratios."

Rose pots 4x9 in a flat, Instructions to students:

  • Use tape to label pots (Section #, Student Pair #, Date, Name)
  • Fill 1/3 with potting soil (contain activity inside soil bucket)
  • add 8 fertilizer pellets, do not mix
  • fill to the top with soil (work inside soil bucket, do not mix)
  • push down soil
  • put pot in mesh tray in sink, hold down
  • add water from a cup to settle the soil
  • put up to 5 seeds on soil surface (hoping for min 1 plant/pot)
  • cover with dusting of soil
  • put in new tray that contains a mesh insert

Instruction to TAs:

  • keep (or place) mesh tray of all collected pots in the sink
  • water gently, carefully ensuring seeds remain at the top surface of the soil
  • ensure soil is packed and moist enough for seeds to germinate
  • when different seed genotypes are used, take care not to transfer any seeds between pots
  • after excess drains into sink, move into a tray and cover with clear dome lid
  • carry tray to growth chamber

Grow in growth chamber in ISB373: 24 hour light, 24C

Water from the bottom (into the tray, not into the pots), as needed

Wisconsin Fast Plants

Week 04 F1 Seedling observations

Week 04 F1 Seedling observations kdorfman Mon, 07/24/2023 - 16:34

Fast Plants Labs week of 9/23/23

(See also micropipetting and yeast DNA extraction)

See the stock document about observations

Transfer inner slotted tray to a dry flat before transport to classroom

All plants should all be purple stem, green leaves

See P1 we planted earlier

Make a prediction about P2

Moodle doc "Fast Plants Observations After One Week" has questions for students while they look at plants.

Week 05 Pollination

Week 05 Pollination kdorfman Mon, 07/24/2023 - 16:46

Fast Plant Labs week of 10/2/23

See also

F1 x F1 pollination

Remove the rare non-purples before class!

(Not the parents we planted!)

Everyone pollinates every plant. (Staff repeats afterward for complete mixing of pollen)

Non-sterile cotton swab.

Week 06 - no labs

Week 06 - no labs kdorfman Wed, 08/09/2023 - 19:50

No Labs - indigenous peoples day

Lab makeups as needed

Week 07 & 08 Plant observations

Week 07 & 08 Plant observations kdorfman Mon, 07/24/2023 - 16:46

Fast Plant Labs weeks of 10/16 and 10/23 2023

(See also Yeast DNA sequencing, )

Observe F2 seedlings

Enter F2 data by pair

Week 10 F2 observations

Week 10 F2 observations kdorfman Mon, 07/24/2023 - 16:55

Fast Plants labs week of 11/6

Plant Data Entry Sheet

See also Prep MC1R for sequencing; gel

Week 12 - no labs

Week 12 - no labs kdorfman Wed, 08/09/2023 - 19:51

11/20 - week of Thanksgiving

Week 13 Plant reports due

Week 13 Plant reports due kdorfman Mon, 07/24/2023 - 16:57

Turn in Fast Plants report week of 11/27/23

Turn in report on Fast Plant genetics experiment

See also Dog genes bioinformatics

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


  • 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


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


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)

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