Protocol 23038: Standard PCR Assay - Ntf3<tm1Jae>

STOCK Ntf3^tm1Jae/J

Stock No: 002276

Version 1.3

Notes

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

Mutant = 330 bp
Heterozygote = 250 bp and 330 bp
Wild type = 250 bp

Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
oIMR0130		CCT GGC TTC TTT ACA TCT CG		Common	A
oIMR0131		TGG AGG ATT ATG TGG GCA AC		Wild type	A
oIMR1694		GGG AAC TTC CTG ACT AGG GG			A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
oIMR0130	0.50 uM
oIMR0131	0.50 uM
oIMR1694	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
002275	B6.129S4-Ntf3^tm1Jae/J
002276	STOCK Ntf3^tm1Jae/J
2 strains use this protocol