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Notes
The way this breeding is done – we get three different phenotypes - db homs are fat and black, Dock 7 or misty homs are lean with a misty coat color & hets are black and lean. So, based on eliminating the two hom classes by phenotype, the only animals that will be genotyped/breeder confirmed are double hets, as we see in all these reqs- From Marge
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Mutant = 200 bp
Heterozygote = 200 bp and 481 bp
Wild Type = 481 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 8895 | GCT GCA GAA TGG ACG GTT GA | Common | A, B | |||
| 8896 | GCA GTG CAC AGG CTC AGG AA | Wild type Reverse | A | |||
| 8897 | AGC CAC TAC AAT CCA CCC CTT G | Mutant Reverse | B |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 8895 | 0.50 uM |
| 8896 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Reaction B
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 8895 | 0.50 uM |
| 8897 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 002048 | B6 x C57BLKS-Dock7m Leprdb Myo15sh2-J/J |
| 000699 | B6.Cg-Dock7m Leprdb/+ +/J |
| 005089 | B6.Cg-Qkiqk-2J/GrsrJ |
| 000027 | B6.D-Tyrp1b Dock7m/J |
| 009659 | B6.D2(BKS)-Dock7m/J |
| 004949 | BKS(Cg)-Grxcr1pi-3J/J |
| 000642 | BKS.Cg-Dock7m +/+ Leprdb/J |
| 000700 | BKS.Cg-Dock7m Leprdb/+ +/J |
| 001049 | BKS.Cg-mea2J Dock7m/+ +/J |
| 001192 | BKS.Cg-meaJ Leprdb +/+ + Dock7m/J |
| 000707 | CBA.Cg-Dock7m Leprdb/+ +/J |
| 11 strains use this protocol | |