ADE1

ade1

ADE2

ade2

Link to Kathleen's photo gallery

Frozen cell lines

Genes

MC1R Alleles

Agouti Alleles

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

Dog DNA labs week of 10/16/23

Instructions for swabbing dog's mouth

Tell students "Go home and swab your dog"

Dog DNA labs, week of 10/23/23

Isolate DNA

1 sample per pair, from:

• Dog cell cultures from Kathleen
• Student dog mouth swabs
• DNA from Cornell BioBank
• Staff dog mouth swabs

QIAamp DNA minikit 51306

Equipment

• 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

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

Equipment

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

Reagents

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

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

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:

• Aci1
• Mnl1

We provide uncut

Bioinformatics

• 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

Thanksgiving week of 11/20/23

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

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

Individual completion of dog Practical

Here are some resources for growing and working with Fast Plants

Purple stem/yellow green leaf stock document

Fast Plants Growing Instructions from Carolina

Fast Plants Care

UWisc fast plants tips

Fast Plants life cycle

Seed to seed in 48 days (video)

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

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.

Fast Plant Labs week of 10/2/23

See also

• yeast DNA sequencing
• Dog DNA extraction

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.

No Labs - indigenous peoples day

Lab makeups as needed

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

(See also Yeast DNA sequencing, )

Observe F2 seedlings

Enter F2 data by pair

Fast Plant Labs week of 10/30/23

See planting instructions

See also Dog DNA quantification

Each person does 2 pots

Fast Plants labs week of 11/6

Plant Data Entry Sheet

See also Prep MC1R for sequencing; gel

Fast Plant labs week of 11/13/23

(See also dog TRYP1 locus, analysis of MC1R genotype & sequence, cap3 sequence assembly)

Compile & analyze data

11/20 - week of Thanksgiving

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

Turn in report on Fast Plant genetics experiment

See also Dog genes bioinformatics

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)

LiCl/KAc

1 part 5M KAc : 2.5 parts 6M LiCl

T4 DNA Ligase

NEB M0202S

Get from New England Biolabs Freezer Program in Fernald

Store in freezer

Comes with 10X ligase buffer (includes ATP

3M NaOAc

PBS

~200 mL/prep

4 preps per group

Aliquot 1 mL per group

Solution A

100 mM Tris-HCl pH 7.5

100mM EDTA

100 mM NaCl

0.5% SDS

Make 10 mL

pH to 7.5

Equipment

• razor blades
• transilluminators
• face shields
• cutting boards
• dry bath at 50C
• columns & collection tubes

Solutions

• Add ethanol to Buffer PE
• Buffer QG ~2 mL (steps 4 & 8)
• isopropyl - ~0.5 mL
• NaOAc 3M, pH 5.0 ~25 µL
• Buffer PE ~ 1.75 mL
• Buffer EB ~ 75 µL (=Tris-Cl 10 mM pH 8.5)
• 6x loading dye
• 6 gels 1.5% (= 300 mL TAE + 4.5 g agarose + 3 µL EtBr)

QIAquick Gel Extraction Kit

(cat. nos. 28704 and 28706) store at room temperature (15°–25°C) for a year

www.qiagen.com/handbooks.

Notes before starting

• The yellow color of Buffer QG indicates a pH7.5.
• Add ethanol (96%–100%) to Buffer PE before use (see bottle label for volume).
• Isopropanol (100%)
• heating block or water bath at 50°C
• All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a microcentrifuge.

Protocol

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 µL). (For >2% agarose gels, add 6 volumes Buffer QG.)

3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel.

4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2 mL collection tube or into a vacuum manifold.

7. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800µL, load and spin/apply vacuum again.

8. If the DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 0.5 mL Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.

9. To wash, add 0.75 mL Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube. Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt-ended ligation), let the column stand 2-5 min after addition of Buffer PE.

10. Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 min at 17,900g (13,000 rpm) to remove residual wash buffer.

11. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.
12. To elute DNA, add 50 µL Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
13. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Get Ape (A Plasmid Editor) for Mac to read the sequences (if it isn't installed already)

http://biologylabs.utah.edu/jorgensen/wayned/ape/