Protocol 29934: QPCR Assay - Human TARDBP

STOCK Tg(Prnp-TARDBP)4Jlel/J

Stock No: 016144

Version 3.0

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

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
10280		GTA CGG GGA TGT GAT GGA TG		Transgene Forward	A
10281		CGC AAT CTG ATC ATC TGC AA		Transgene Reverse	A
10282	Fluorophore-1	CCA AGC CAT TCA GGG CCT TTG C	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
10280	0.40 uM
10281	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
010700	B6.Cg-Tg(Prnp-TARDBP*A315T)95Balo/J
017933	B6.Cg-Tg(Prnp-TARDBP*Q331K)103Dwc/J
028413	B6;C3-Tg(NEFH-tTA)8Vle Tg(tetO-TARDBP)12Vle/J
016841	B6;C3-Tg(tetO-TARDBP)12Vle/J
017930	B6N.Cg-Tg(Prnp-TARDBP*Q331K)109Dwc/J
016608	C57BL/6-Tg(Prnp-TARDBP)3cPtrc/J
017604	C57BL/6-Tg(Prnp-TARDBP*M337V)4Ptrc/J
016144	STOCK Tg(Prnp-TARDBP)4Jlel/J
8 strains use this protocol