Stock No: 000051
Protocol 30198: Separated PCR Assay - A<w-J> A<y> A<w>
Version 6.0

Notes

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

A = 763 bp (and 162 in C3H A allele)


a (nonagouti/black) = 839 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR7876 GGC CAC ACT TAG GGA GTT CA Common A, B
oIMR7932 GGT TGG CCA CCA TGT CTA GT Mutant Reverse B
oIMR9013 TGC CTT ATT CTG TTT TTG TTC C Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR7876 0.50 uM
oIMR9013 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
oIMR7876 0.50 uM
oIMR7932 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
002016 B6(Cg)-Aw-J EdaTa-6J Chr YB6-Sxr/EiJ
100409 B6129PF1/J-Aw-J/Aw
000314 B6CBACa Aw-J/A-EdaTa/J-XO
002703 B6CBACa Aw-J/A-Hydinhy3/J
000287 B6CBACa Aw-J/A-Plp1jp EdaTa/J
002343 B6EiC3Sn a/A-Otcspf/J
000200 C3FeB6 A/Aw-J-Ankank/J
000569 C57BL/6J-Aw-J-EdaTa +/+ ArTfm/J
000051 C57BL/6J-Aw-J/J
002468 KK.Cg-Ay/J
001432 STOCK a/a Tyrp1b Ndc1sks/Tyrp1b +/J
11 strains use this protocol