Protocol 30021: QPCR Assay - Tg(FXN)
Version 4.0

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
9693 GAA CTT CAA ATT AGT TCC CCT TTC TTC Forward A
9694 CAC AGC CAT TCT TTG GGT TTC Reverse A
9695 Fluorophore-1 ATC TGA CCC AGT TAC GCC ACG GC 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
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
9693 0.40 uM
9694 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
012253 B6.Cg-Fxntm1Mkn Tg(FXN)YG8Pook/J
031007 STOCK Fxnem2.1Lutzy Tg(FXN)Y47Pook/J
030395 STOCK Fxnem2.1Lutzy Tg(FXN)YG8Pook/800J
030930 STOCK Fxnem2.1Lutzy Tg(FXN)YG8Pook/J
024097 STOCK Fxntm1Mkn Tg(FXN)Y47Pook/J
024113 STOCK Fxntm1Mkn Tg(FXN)YG8Pook/2J
008398 STOCK Fxntm1Mkn Tg(FXN)YG8Pook/J
030324 STOCK Tg(FXN)YG8Pook/J
8 strains use this protocol