Dog Genetics Labs 2023-F
Dog Genetics Labs 2023-F kdorfman Thu, 07/20/2023 - 18:08Here are the prep pages for the dog genetics labs 2023
Puritan Try Cap-shure Swabs with attached caps from McKesson
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
Dog DNA summary
Dog DNA summary kdorfman Wed, 08/02/2023 - 14:47Genes
| 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 |
|---|---|---|---|
| E | |||
| ee |
Agouti Alleles
| letter | gene | full name | traits |
|---|---|---|---|
| Ay | |||
| Aw | |||
| aa |
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
Agouti sequences
Agouti sequences kdorfman Mon, 08/21/2023 - 14:08ASIP
To find the crucial 82, 83, 96 amino acids:
_ _ PRPP...CVAT _
82 83 ................. 96
MC1R sequences
MC1R sequences kdorfman Mon, 08/21/2023 - 14:10MC1R
TYRP sequences
TYRP sequences kdorfman Mon, 08/21/2023 - 14:09TyRP
defB103
defB103 kdorfman Mon, 08/21/2023 - 14:10defB
Week 07 DNA collection
Week 07 DNA collection kdorfman Mon, 07/24/2023 - 21:00Dog 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 - 20:25Dog 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
DNA from cultured cells
DNA from cultured cells kdorfman Tue, 08/22/2023 - 16:04From 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.
Procedure
- Harvest cells
- Trypsinize and count cells
- Put no more than 5 million cells into a microfuge tube
- Centrifuge for 5 min at 300 x g.
- Remove the supernatant completely and discard, taking care not to disturb the cell pellet.
- Resuspend cell pellet in PBS to a final volume of 200μL.
- Add 20 μL QIAGEN Protease or proteinase K.
- (Continue with step 3 of “Protocol: DNA Purification from Blood or Body Fluids (Spin Protocol)”, page 27)
Week 09 PCR MC1R
Week 09 PCR MC1R kdorfman Mon, 07/24/2023 - 20:25Dog 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
Week 10 Exo-SAPiT
Week 10 Exo-SAPiT kdorfman Mon, 07/24/2023 - 20:27Dog 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 - 20:27Dog 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
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
Week 12 - no labs
Week 12 - no labs kdorfman Wed, 08/09/2023 - 19:56Thanksgiving week of 11/20/23
Week 13
Week 13 kdorfman Mon, 07/24/2023 - 20:27Dog 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 - 20:29Dog genotype & phenotype presentations, week of 12/4/23 (last week of classes)
Individual completion of dog Practical