Protocol 15154: Pyrosequencing Assay - Kit<W>

WB/ReJ Kit^W/J

Stock No: 000692

Version 4.0

Notes

This genotyping assay uses pyrosequencing technology and is run on the Biotage PSQ 96MA. The Jackson Laboratory is not posting the complete details of our pyrosequencing genotyping assays as the primers for pyrosequencing cannot be used for sequencing using more traditional methods. The wild type and mutant nucleotides and the flanking DNA sequence are provided below.

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 = T/T
Heterozygote = C/T
Wild type = C/C

Sequencing primer is on the reverse strand.

Sequence

AGCAAATCCAGGCCCACACTCTGTTCACGCCGCTGCTCATTGGCTTTGTGGTTG

CAGCTGGCGCGATGGGGATCATTGTGATGGTGCTCACCTACAAATATTTGCAG(g/a)tg

agcattgaattgttctcttcctggggacgccaaggcggcagggcaggcactgattgt

tcagcggg

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
poIMR0178	Fluorophore	GGG GAT CAT TGT GAT GGT GCT
poIMR0179		CCC CAG GAA GAG AAC AAT TCA A
poIMR0180		AGA ACA ATT CAA TGC TCA

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
poIMR0178	0.50
poIMR0179	0.50
Glycerol	5.00
Kapa 2G HS taq polym	0.01
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
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.

Strains Using This Protocol

Stock Number	Strain Name
000164	B6.Cg-Kit^W/J
000692	WB/ReJ Kit^W/J
2 strains use this protocol