Genotyping Protocols Database
Stock No: 002726
Protocol 30009: QPCR Assay - Sod TgN Copy Number
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 Fuorophore/Quencher combination.  The transgene genotype is determined by comparing ΔCt values of each unknown sample against known high copy 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
13854 Fluorophore-1 CTG CAT CTG GTT CTT GCA AAA CAC CA 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
oIMR9665 GGG AAG CTG TTG TCC CAA G Transgene A
oIMR9666 CAA GGG GAG GTA AAA GAG AGC Transgene 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
oIMR1544 0.40 uM
oIMR3580 0.40 uM
oIMR9665 0.40 uM
oIMR9666 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
002299 B6.Cg-Tg(SOD1*G93A)dl1Gur/J
002726 B6SJL-Tg(SOD1*G93A)1Gur/J
002994 B6.129X1-Baxtm1Sjk/J
008229 B6.Cg-Tg(SOD1*G37R)29Dpr/J
013199 FVB.Cg-Tg(SOD1*G93A)1Gur/J
016149 B6.Cg-Tg(SOD1*G37R)1Dwc/J
6 strains use this protocol