Protocol 30018 - Tg(FCGRT)-qPCR

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
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
ddH2O
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
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
021146	B6.Cg-Fcgrt^tm1Dcr Prkdc^scid Tg(CAG-FCGRT)276Dcr/DcrJ
018441	B6.Cg-Fcgrt^tm1Dcr Prkdc^scid Tg(FCGRT)32Dcr/DcrJ
004919	B6.Cg-Fcgrt^tm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ
014565	B6.Cg-Fcgrt^tm1Dcr Tg(FCGRT)32Dcr/DcrJ
029686	B6.Cg-Tg(FCGRT)32Dcr Fcgrt^tm1Dcr Ighg1^{em2(IGHG1)Mvw}/MvwJ
025099	BXSB.Cg-Fcgrt^tm1Dcr Tg(FCGRT)99Dcr/J
6 strains use this protocol