Genotyping Protocols Database
Stock No: 004194
Protocol 22188: QPCR Assay - Tg(Tcrb)425Cbn QPCR
Version 5.0

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

Primers in this protocol are not specific for the transgene.  WT samples will have PCR product but will have a lower copy number.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10102 Fluorophore-1 CAG CAG ATT CTC AGT CCA ACA G Quencher-1 Transgene Forward A
10103 Fluorophore-2 GCA CAG AAG TAC ACG GCA GA Quencher-2 Transgene Reverse A
13646 Fluorophore-3 CTC TAG CTC CAA GGC ACT CAT GTT CAT CTC A Quencher-3 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-4 CCA ATG GTC GGG CAC TGC TCA A Quencher-4 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
10102 0.40 uM
10103 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

This is the only strain that uses this protocol.