Protocol 30018: QPCR Assay - Tg(FCGRT)-qPCR
Version 3.0


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
10931 GCC ATT CCG ACT GCT AAA AG Transgene Forward A
10932 AGA TGC CAG TGT TCC AGG AG Transgene Reverse A
10933 Fluorophore-1 AGT CAG GCC CCT TTC ATG CTG TGA 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
Kapa Probe Fast QPCR 1.00 X
10931 0.40 uM
10932 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
Tg Probe 0.15 uM
IC Probe 0.15 uM


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
021146 B6.Cg-Fcgrttm1Dcr Prkdcscid Tg(CAG-FCGRT)276Dcr/DcrJ
018441 B6.Cg-Fcgrttm1Dcr Prkdcscid Tg(FCGRT)32Dcr/DcrJ
004919 B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ
014565 B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr/DcrJ
029686 B6.Cg-Tg(FCGRT)32Dcr Fcgrttm1Dcr Ighg1em2(IGHG1)Mvw/MvwJ
025099 BXSB.Cg-Fcgrttm1Dcr Tg(FCGRT)99Dcr/J
6 strains use this protocol