Stock No: 008468
Protocol 13657: QPCR Assay - Tg(tetO-DTA)1Gfi
Version 5.0

Notes

Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler 480. 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
oIMR1544 CAC GTG GGC TCC AGC ATT
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G
oIMR8938 AGA GCG GGT TTT CAT TAT CTA CA
oIMR8939 TAC ACA AGG AAA TTA TGA CGA TGA
TmoIMR0105 Fluorophore-1 CCA ATG GTC GGG CAC TGC TCA A Quencher-1
TmoIMR0125 Fluorophore-2 CCG CAG CGT CGT ATT TAT TGT CGG T Quencher-2

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.91
ddH2O
oIMR8938 0.43
oIMR8939 0.43
oIMR1544 0.00
oIMR3580 0.00
TmoIMR0125 1.27
TmoIMR0105 0.00
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
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
008468 B6.Cg-Tg(tetO-DTA)1Gfi/J
008755 STOCK Tg(Ins2-rtTA)2Efr Tg(teto-DTA)1Gfi/J
008168 STOCK Tg(tetO-DTA)1Gfi/J
3 strains use this protocol