Protocol 30056: QPCR Assay - Tg(Ins2-OVA)
Version 5.0

Notes

Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler
480. 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
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
oIMR6419 CCA TTT GCC AGT GGG ACA AT Transgene A
oIMR6420 TCA AGC TGC TCA AGG CCT G Transgene A
TmoIMR0086 Fluorophore-1 CAT GTT GGT GCT GTT GCC TGA TGA AGT C Quencher-1 Tg Probe
TmoIMR0107 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
oIMR1544 0.40 uM
oIMR3580 0.40 uM
oIMR6419 0.40 uM
oIMR6420 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
005432 C57BL/6-Tg(Ins2-OVA)307Wehi/WehiJ
005433 C57BL/6-Tg(Ins2-OVA)59Wehi/WehiJ
005431 C57BL/6-Tg(Ins2-TFRC/OVA)296Wehi/WehiJ
3 strains use this protocol