Protocol 24041: Separated PCR Assay - H2-Ab1<tm1Gru>alternate2
Version 1.2

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

Mutant = ~295 bp
Heterozygote = 185 bp and ~295 bp
Wild type = 185 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
15442 ATG GGC GAG TGC TAC TTC AC Wild type Forward B
15801 GCC CGC GTT CGC TCC AGG Common A, B
oIMR1100 GCT ATC AGG ACA TAG CGT TGG Mutant Forward A neo F

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
15801 0.50 uM
oIMR1100 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
15442 0.50 uM
15801 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
002956 B6NTac.Cg-H2-Ab1tm1Gru Tg(Ab1TL)1Gru/J
004256 NOD.129S2(B6)-H2-Ab1tm1Doi/DvsJ
006021 NOD.Cg-H2-Ab1tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ
006023 NOD.Cg-H2-Ab1tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ
021885 NOD.Cg-Prkdcscid H2-Ab1tm1Doi Il2rgtm1Wjl/SzJ
026561 NOD.Cg-Prkdcscid H2-Ab1tm1Doi Il2rgtm1Wjl Tg(HLA-DQA1,HLA-DQB1)1Dv/SzJ
006024 NOD.Cg-Rag1tm1Mom H2-Ab1tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ
006022 NOD.Cg-Rag1tm1Mom H2-Ab1tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ
030331 NOD.Cg-Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz Prkdcscid H2-Ab1tm1Doi Il2rgtm1Wjl/SzJ
9 strains use this protocol