Protocol 24926: Standard PCR Assay - Generic Human FXN
Version 2.2

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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

Melt Peaks
Wt = 82.3°C +/- 1
Tg = 86.3°C +/- 1


Transgene = 595 bp
Internal positive control = 324 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11820 GCA AAA GCT GGA GAT CAA AGT GTG A Transgene A FXN
11821 GAG GCA ACA CAT TCT TTC TAC AGA Transgene A FXN
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Internal Positive Control A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
11820 0.50 uM
11821 0.50 uM
oIMR7338 0.50 uM
oIMR7339 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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 (Touchdown)
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.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
008586 B6.Cg-Tg(FXN*)1Sars Fxntm1Mkn/J
031007 STOCK Fxnem2.1Lutzy Tg(FXN)Y47Pook/J
024113 STOCK Fxntm1Mkn Tg(FXN)YG8Pook/2J
010963 STOCK Fxntm1Mkn Tg(FXN)YG22Pook/J
018299 STOCK Tg(FXN*)1Sars Fxntm1Mkn/J
5 strains use this protocol