Protocol 23440: Standard PCR Assay - Fxn<tm1Pand> GAA repeats

B6.Cg-Fxn^tm1.1Pand Fxn^tm1Mkn/J

Stock No: 012329

Version 1.2

Notes

AM Bufer:  670 mM TrisHCl pH 8.8, 166 mM (NH4)2SO4, 20 mM MgCl2, 1.7 mg/ml BSA, 10 mM 2-mercaptoethanol (Add 2-mercaptoethanol to 10 mM just prior to use. Do not store with 2-mercaptoethanol in buffer.) THIS PROTOCOL DOES NOT NEED THE MERCAPTOETHANOL ADDED.

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

JR8470, JR11113, JR12329, JR14162 in 900-950 bp range

B6 = no band
human = ~194 bp

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
8579	Fluorophore	GAC TAA CCT GGC CAA CAT GG			A
8580	Fluorophore	CTT GGC TTA ATG CAA CCT CTG			A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTPS-kapa	0.26 mM
8579	0.50 uM
8580	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
011113	B6.129-Fxn^tm1.1Pand/J
008470	B6.129-Fxn^tm1Pand/J
012329	B6.Cg-Fxn^tm1.1Pand Fxn^tm1Mkn/J
008398	STOCK Fxn^tm1Mkn Tg(FXN)YG8Pook/J
4 strains use this protocol