Protocol 22141: QPCR Assay - Generic DsRed

C57BL/6-Tg(Irgm1-DsRed2)1Nci/Mmjax

Stock No: 037280

Version 7.0

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

Tg = 69 bp

IPC= 74 bp

Strain 414192 - results should match Tg(Ins2-HBEGF)6832Ugfm.

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
oIMR4082		TGA AGG GCG AGA TCC ACA A		Transgene	A
oIMR4083		TGG ACT TGA ACT CCA CCA GGT A		Transgene	A
TmoIMR0067	Fluorophore-1	CTG AAG CTG AAG GAC GGC GGC C	Quencher-1	Tg Probe
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
ddH2O
oIMR1544	0.40 uM
oIMR3580	0.40 uM
oIMR4082	0.40 uM
oIMR4083	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
040785	B6(Cg)-Notch3^tm1.1(NOTCH3)Arbo/J
006055	B6.Cg-Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J
006051	B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J
008705	B6.Cg-Tg(CAG-DsRed,-EGFP)5Gae/J
006872	B6.Cg-Tg(Ins1-DsRed*T4)32Hara/J
018410	B6.Cg-Tg(Pnkd*A7V*A9V,-DsRed)671Ljp/J
040890	B6.FVB-Tg(Hcar2-mRFP)1Soff/J
013082	B6.FVB-Tg(Per2-DsRed*T3)12Obr/Mmjax
006679	B6;129P2-Or8a1^tm27Mom/MomJ
008605	B6;C3-Tg(CAG-DsRed,-EGFP)5Gae/J
029332	B6N.Cg-Tg(P2rx4-tdTomato)1Khakh/J
031160	C.FVB-Tg(Acta2-DsRed)1Rkl/J
024046	C57BL/6-Tg(Csf1r-HBEGF/mCherry)1Mnz/J
037280	C57BL/6-Tg(Irgm1-DsRed2)1Nci/Mmjax
040416	C57BL/6J-Igs2^em1(CAG-mCherry,-Cxcr3*,-GFP*)Miji/J
040208	C57BL/6J-Igs2^em1(CAG-RFP*/GFP*)Qsun/J
009655	C57BL/6J-Tg(Dcx-DsRed)14Qlu/J
005328	NOD/ShiLt-Tg(Cd4-DsRed)4Lt/J
039157	STOCK Lcn2^tm2Bara Tyr^c-2J/J
040810	STOCK Pou2af1^em1Dtan/J
005438	STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J
005441	STOCK Tg(CAG-DsRed*MST)1Nagy/J
025693	STOCK Tg(CAG-mCherry/Pxn)#Mgin/KleyJ
008241	STOCK Tg(Cspg4-DsRed.T1)1Akik/J
006866	STOCK Tg(Ins1-DsRed*T4)32Hara/J
25 strains use this protocol