Protocol 30954: Probe Assay - Generic Human FXN
Version 1.0

Notes

This assay cannot distinguish hemi from hom

 

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

Tg=  70 bp

IPC = 74 bp

Sequence

Tg Sequence:

 

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35841 TTG TGG AAT CAG TGG TTC ATC T Transgene Forward A
35842 CAC TGG AAG ACA AAA CAA ATG C Transgene Reverse A
35843 Fluorophore-1 AAG GGC TGT GCT GTG GAA TTA 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
35841 0.40 uM
35842 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 4.0 -- Forever
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
030930 STOCK Fxnem2.1Lutzy Tg(FXN)YG8Pook/J
024097 STOCK Fxntm1Mkn Tg(FXN)Y47Pook/J
030324 STOCK Tg(FXN)YG8Pook/J
5 strains use this protocol