Genotyping Protocols Database
Protocol 21556: QPCR Assay - Tg(HLA-A)-2
Version 7.0

Notes

 

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate
instrument specific Fluorophore/Quencher combination. The transgene genotype is
determined by comparing ΔCt values of each unknown sample against known
homozygous and hemizygous controls, using appropriate endogenous references.

 

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

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11287 GGA GAC ACG GAA AGT GAA GG Transgene Forward A
11288 GGC CTC GCT CTG GTT GTA G Transgene Reverse A
11289 Fluorophore-1 CCG AGT GGA CCT GGG GAC CC Quencher-1 Tg Probe
oIMR1544 CAC GTG GGC TCC AGC ATT Internal Positive Control Forward A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe

Reaction A

Component Final Concentration
ddH2O
Kapa Probe Fast QPCR 1.00 X
11287 0.40 uM
11288 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
Tg Probe 0.15 uM
IC Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 -- repeat steps 2-3 for 40 cycles
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
003475 C57BL/6-Mcph1Tg(HLA-A2.1)1Enge/J
005512 NOD.B6-Mcph1Tg(HLA-A2.1)1Enge/DvsJ
006611 NOD.Cg-B2mtm1Unc Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ
009617 NOD.Cg-Mcph1Tg(HLA-A2.1)1Enge Prkdcscid Il2rgtm1Wjl/SzJ
006609 NOD.Cg-Mcph1Tg(HLA-A2.1)1Enge Prkdcscid/DvsJ
014570 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A/H2-D/B2M)1Dvs/SzJ
033127 NOD.Cg-Rag1tm1Mom Flt3tm1Irl Mcph1Tg(HLA-A2.1)1Enge Il2rgtm1Wjl/J
006604 NOD/ShiLtDvs-Tg(HLA-A/H2-D/B2M)1Dvs/J
8 strains use this protocol