Protocol 13299: QPCR Assay - Tg(CAG-DsRed*MST)1Nagy
Version 2.1

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
oIMR1544 CAC GTG GGC TCC AGC ATT
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G
oIMR4082 TGA AGG GCG AGA TCC ACA A
oIMR4083 TGG ACT TGA ACT CCA CCA GGT A
TmoIMR0067 Fluorophore-1 CTG AAG CTG AAG GAC GGC GGC C Quencher-1
TmoIMR0107 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2

Reaction A

Component Final Concentration
ddH2O
Kapa Probe Fast QPCR 1.91
oIMR4082 0.43
oIMR4083 0.43
oIMR1544 0.00
oIMR3580 0.00
TmoIMR0067 1.27
TmoIMR0107 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

This is the only strain that uses this protocol.