Protocol 14597: Pyrosequencing Assay - Tyr c

C.C3-Tlr4^Lps-d/J

Stock No: 002930

Version 2.0

Notes

TGS Design to targeted SNP, JAK 4/19/11.  No SNPs present in primers, UCSC PCR in silico 4/19/11.
Amplicon length  = 122 bp
Score = 95
ID 1

This mutation introduces a DdeI enzyme restriction site.

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

Sequence

ATGTTCTTGGCTGTTTTGTATTGCCTTCTGTGGAGTTTCCAGATCTCTGATGGCCATTTTCCTCGAGCCTGTGCCTCCTCTAAGAACTTGTTGGCAAAAGAATG
CTGCCCACCATGGATGGGTGATGGGAGTCCCTGCGGCCAGCTTTCAGGCAGAGGTTCCTGCCAGGATATCCTTCTGTCCAGTGCACCATCTGGACCTCAGTTCCCCTTCAAAGGGGTGGA
TGACCGTGAGTCCTGGCCCTCTGTGTTTTATAATAGGACCTGCCAGTGCTCAGGCAACTTCATGGGTTTCAACTGCGGAAACT(g/c)TAAGTTTGGATTT
GGGGGCCCAAATTGTACAGAGAAGCGAGTCTTGATTAGAAGAAACATTTTTGATTTGAGTGTCTCCGAAAAGAATAAGTTCTTTTCTTACCTCACTTTAGCAAAACATACTATCAGCTCAGTCT
ATGTCATCCCCACAGGCACCTATGGCCAAATGAACAATGGGTCAACACCCATGTTTAATGATATCAACATCTACGACCTCTTTGTATGGATGCATTACTATGTGTC

Albino = C

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
12243	Fluorophore	TCC TGG CCC TCT GTG TTT TAT A
12244		ATC AAG ACT CGC TTC TCT GTA CAA
12245		CCC CAA ATC CAA ACT T

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
12243	0.50
12244	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
002930	C.C3-Tlr4^Lps-d/J
002106	KK/HlJ
2 strains use this protocol