Protocol 23418: Standard PCR Assay - Tg(FXN)YG8Pook GAA Repeat
Version 3.2

Notes

The Fine Mapping Lab uses an oIMR8596R primer that is fluorescently tagged with FAM on the 5' end.

Size on ABI 3730.
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

C57BL/6 = no band, human band approximately 480 bp. 

Stock# 012963 = 2-3 bands from ~1000bp -1100 bp
Stock# 008398 = 3-4 bands form ~810 bp -1050 bp
Stock# 012910 = 2-3 bands from ~1070 bp - 1300 bp
 

JR 24097 contains 9 GAA repeats

JR 24113 contains ~200 GAA repeats

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8595 GGG ATT GGT TGC CAG TGC TTA AAA GTT AG Forward A
oIMR8596 GAT CTA AGG ACC ATC ATG GCC ACA CTT GCC Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR8595 0.50 uM
oIMR8596 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
012910 B6.Cg-Fxntm1Mkn Tg(FXN)YG22Pook/J
024113 STOCK Fxntm1Mkn Tg(FXN)YG8Pook/2J
008398 STOCK Fxntm1Mkn Tg(FXN)YG8Pook/J
3 strains use this protocol