Protocol 22206: QPCR Assay - Tg(tTA) qPCR

STOCK Tg(Camk2a-tTA)1Mmay Fgf14^{Tg(tetO-MAPT*P301L)4510Kha}/J

Stock No: 024854

Version 3.0

Notes

Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler 480. Use an appropriate instrument-specific Fuorophore/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.

Primer 9344 replaced with 13639, 3' Iowa Black with Zen

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
13734	Fluorophore-1	CGC ATG CCG ACG CGC TAG	Quencher-1	Tg Probe
9342		GGA CGA GCT CCA CTT AGA CG		Transgene Forward	A
9343		CAA CAT GTC CAG ATC GAA ATC		Transgene Reverse	A
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
9342	0.40 uM
9343	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
016198	129S6.Cg-Tg(Camk2a-tTA)1Mmay/JlwsJ
007004	B6.Cg-Tg(Camk2a-tTA)1Mmay/DboJ
038301	B6;SJL-Tg(ACTA1-rtTA)6Mcrth/J
005734	NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ
006999	STOCK Dbt^tm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J
024854	STOCK Tg(Camk2a-tTA)1Mmay Fgf14^Tg(tetO-MAPT*P301L)4510Kha/J
003273	STOCK Tg(CMV-rtTA)4Bjd/J
7 strains use this protocol