Protocol 13305: Pyrosequencing Assay - Hydin<hy3>

B6CBACa A^w-J/A-Hydin^hy3/J

Stock No: 002703

Version 2.1

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 = 3 C's
Wild type = 4 C's

Sequence

AACCTCCACGACTAAATGCATGATAAATACTTTAGTTAAGTCATGAATGCATGAAGTGT
ATGTGTTGGTGTATGACTGGGGATTTAAGGAGCTAACAGGGAATGGATATAAGCAGAG
GGAATGACACACATCTGTGAAGATAAAGATGTCACAGTGAGACCCTAGTTTGTATATA
CATTAAACATAGTTTTAAAGAGGGCCTGGGGATGAGGCTGAGACACTGAAGTGCCAG
GAGGGCAATGGAACCTGAGATTGATCGACCGGAACCCACGTGAACTATCTGGACGCA
GTGGGGCATACTTAAGATACCAAATTTGGAAAGGGAGAGAGACAGGGGTCCTTGAGT
TTTGCTGGATATCCAGTTTCCCCCAACCAGGAAACCCCAGGCTCAGTGAAAGATCCT
GAATCAAAAAACTAAAGTGTATAGAGGAAAACACCTGATATTGAGCTCTGACCTGTA
CGCATACACCCACCACATTAAATAAATAAATAAATAAATAAATAAATAAATAAATAAAT
AAATAAAATACACACATACACATGCATGCACATGCATACACATACATACATATACATAC
ATATAAATACATATACATACATACTTATTTACTTACTTACTTAAGACAAGTTAGAAAGTA
ATGAGACATAGTAATGCATTATATATACAATCCTAAGTCATTGCCTTAGGATTTGTTGAT
CAAGGGAGGCTACTGGGGGAAAGAAGTCTTTCTCACCTCAAATTTTATCTTCCAGGA
TTT(cccc)ATACCTTGATGTGTTCCCTCAATAATACATCTTTGGTCCCCATGACCTTCAAA
CTGCGTGTCCGCGGCGATGGCGAAGGCATGAGTAGCATCCCAAGTTACTCTCAGGAG
TCAGACAGCAAGCAATGGTCCGGGATTAATACAGAAATGCCTACCACAAAACCCAAA
GAATTCACCATCTCTCCCAACAGTGGCACCATCCGTGCCCAGGGCTTTACTGCCATCAAGGTAAAGTGT

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
poIMR0272		GTC ATT GCC TTA GGA TTT GTT GAT
poIMR0273	Fluorophore	CAT GGG GAC CAA AGA TGT ATT ATT
poIMR0274		CTT CCA GGA TTT CCC

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
poIMR0272	0.50
poIMR0273	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
002703	B6CBACa A^w-J/A-Hydin^hy3/J
008343	FVB.Cg-Hydin^hy3/MlrJ
2 strains use this protocol