Stock No: 008250
Protocol 22205: QPCR Assay - Tg(tTA)
Version 10.0

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
Kapa Probe Fast QPCR 1.00 X
ddH2O
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
002618 B6.Cg-Tg(MMTVtTA)1Mam/J
034533 B6.FVB-Tg(EmuSR-tTa)83Bop/DwfJ
007678 B6;SJL-Tg(KRT14-rtTA)208Jek/J
003170 FVB.Cg-Tg(Myh6-tTA)6Smbf/J
034802 FVB/N-Tg(MMTV-tTA)25754Kuw/Mmjax
004127 FVB/N-Tg(Nes-rtTA)306Rvs/J
006875 FVB/N-Tg(Tagln-rtTA)E1Jwst/J
004937 NOD.Cg-Tg(Ins2-tTA)1Doi/DoiJ
005734 NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ
006999 STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J
024108 STOCK Igs7tm93.1(tetO-GCaMP6f)Hze Tg(Camk2a-tTA)1Mmay/J
024854 STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J
003272 STOCK Tg(Cebpb-tTA)5Bjd/J
003273 STOCK Tg(CMV-rtTA)4Bjd/J
003271 STOCK Tg(CMV-tTA)3Bjd/J
008250 STOCK Tg(Ins2-rtTA)2Efr/J
016145 STOCK Tg(Scgb1a1-rtTA)2Jaw/J
016146 STOCK Tg(SFTPC-rtTA)2Jaw/J
19 strains use this protocol