Stock No: 008343
Protocol 13305: Pyrosequencing Assay - Hydin<hy3>
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 Aw-J/A-Hydinhy3/J
008343 FVB.Cg-Hydinhy3/MlrJ
2 strains use this protocol