Protocol 24926 - Generic Human FXN

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	Sequence 5' → 3'	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 Fxn^tm1Mkn*/J
031007	STOCK Fxn^em2.1Lutzy Tg(FXN)Y47Pook/J
024097	STOCK Fxn^tm1Mkn Tg(FXN)Y47Pook/J
024113	STOCK Fxn^tm1Mkn Tg(FXN)YG8Pook/2J
010963	STOCK Fxn^tm1Mkn Tg(FXN)YG22Pook/J
008398	STOCK Fxn^tm1Mkn Tg(FXN)YG8Pook/J
018299	STOCK Tg(FXN)1Sars Fxn^tm1Mkn*/J
7 strains use this protocol