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

Tg=   126 bp

IPC = 74 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
27178 AAT CGC ACC CTT CTG TCT TC Transgene Forward A
27179 CAA GGT CCC TCC GAA ACT G Transgene Reverse A
27180 Fluorophore-1 CCA CCA CCT GCC CAC ATC CA Quencher-1 Tg Probe A
oIMR1544 CAC GTG GGC TCC AGC ATT Wild type Forward A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Wild type Reverse A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
27178 0.40 uM
27179 0.40 uM
27180 0.40 uM
oIMR1544 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
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
026936 NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl Tg(HLA-DQA1,HLA-DQB1)1Dv Tg(INS*)172Dvs/DvsJ
2 strains use this protocol