Protocol 36718: End Point Analysis Assay - H2-K1<d-em1Dvs> EP (NOD only)
Version 1.0

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

Mutant = -/-
Heterozygote = G/-
Wild type = G/G

Sequence

Mut CGCTGTCGGCTACGTGGACGACACGCAGTTCGTGCGCTTCGACAGCGACGCGGATAATCCGAGATTTGacCGCGGGCGCCGTGGATGGAGCAGGAGGGGCCGGAGTATTGGGAGGAGCAGACACAGAGAGCCAAGAGCGATGAGCAGTGGTTCCGAGTGAGCCTGA

 

Wt TGTCGGCTACGTGGACGACACGCAGTTCGTGCGCTTCGACAGCGACGCGGATAATCCGAGATTTGAgCCGCGGGCGCCGTGGATGGAGCAGGAGGGGCCGGAGTATTGGGAGGAGCAGACACAGAGAGCCAAGAGCGATGAGCAGTGGTTCCGAGTGAGCCTGAGGAC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49434 GAC AGC GAC GCG GAT AAT Forward A
49435 TCT GCT CCT CCC AAT ACT CC Reverse A
49436 Fluorophore-1 GAG ATT TGA CCG CGG GCG Quencher-1 MUT Probe
49437 Fluorophore-2 GAG ATT TGA GCC GCG GGC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49434 0.40 uM
49435 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
039054 NOD.Cg-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs Tg(HLA-DQA1,HLA-DQB1)1Dv/DvsJ
039056 NOD.Cg-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-DQA1,HLA-DQB1)1Dv Tg(CD2-TcraA1.9,Cd4-TcrbA1.9)1Dvs Tg(INS*)172Dvs/DvsJ
039053 NOD.Cg-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-DQA1,HLA-DQB1)1Dv/DvsJ
036578 NOD.Cg-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs Tg(HLA-DQA1,HLA-DQB1)1Dv Tg(CD2-Tcra20D11,Cd4-Tcrb20D11)1Dvs/DvsJ
039055 NOD.Cg-Prkdcscid H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Il2rgtm1Wjl Tg(HLA-DQA1,HLA-DQB1)1Dv/DvsJ
032927 NOD.Cg-Prkdcscid H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Il2rgtm1Wjl/SzJ
031854 NOD/ShiLt-H2-D1b-em4Dvs/J
031855 NOD/ShiLt-H2-K1d-em1Dvs H2-D1b-em5Dvs/J
031853 NOD/ShiLt-H2-K1d-em4Dvs/J
031433 NOD/ShiLtDvs-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ
031858 NOD/ShiLtDvs-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs/DvsJ
031856 NOD/ShiLtDvs-H2-K1d-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ
031857 NOD/ShiLtDvs-H2-K1d-em1Dvs H2-D1b-em5Dvs Tg(HLA-B39/H2-D/B2M)2Dvs/DvsJ
13 strains use this protocol