Stock No: 004608
Protocol 14219: Pyrosequencing Assay - Htra2<mnd2>
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 = A/T
Wild type = A/A

Sequence

ACTTCTCCCCGGAGCCAGTACAATTTCATCGCAGATGTGGTGGAGAAGACAGCCCCTG
CTGTGGTCTATATCGAGATCCTAGACCGGTAATGGTGAAGTTGACCAGGAGGTGTTGA
ACTGCCGTTAGTGAAGGCAAGGGAGCAGGATGAGGTGGAAGCTGCAGTGTAATGGGT
ACTTTCCCCATCCCAGGCACCCTTTCTCCGGCCGTGAAGTCCCCATCTCAAACGGATC
AGGATTCGTAGTGGCTTCAGATGGGCTCATCGTTACCAACGCCCACGTGGTGGCTGAT
CGGCGCCGAGTACGAGTGAGGCTGCCTAGCGGGGATACTTATGAGGCCATGGTCACA
GCTGTGGATCCCGTAGCAGACATTGCCACACTGAGGATTCAAACCAAGGTGGGGATG
GAGTAGGCCAGATCTGATGGCAGCTGTTCTCTTGCTCCCTTCTTAAAATGACCAGTTTT
GCTGGCCTTGTTTCCCTTAAGGAGCCTCTTCCCACACTGCCCCTCGGCCGCTCTGCTG
ATGTCCGGCAAGGGGAGTTTGTTGTTGCCATGGGAAGCCCCTTTGCACTGCAGAACA
CGATCACATCTGGTATTGTC(a/t)GCTCTGCTCAGCGCCCAGCCAGGGACCTGGGACTC
CCTCAAAACAACGTGGAATACATTCAGACCGATGCAGCTATTGATGTGCGTCCTGATA
TGAGGGAAATTAGGTGCTAAGGTTGGAATTGGGGAGAACAGCTGTTGGGTATGAGCA
CCAACTGACACGTGGTGGTGACTGGGCCTGTATCAGAGGTTGGGCTGCAAAAGGCAG
CCTCTCTAGTTCATGTGCTAGGAATGGAATTTGAAAAATTTCCCTACACCCTGTTTATGT
CCTCGGTCTTAGAATTCCCAGAGCATGGGATTCCAGATCTTTTTGATGTTTTCTTTCATT
TTATCTCACAGTTTGGAAATTCTGGTGGTCCCCTGGTTAACCTGGTGAGTGTCCGTGTTCTTCTAAGAATC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
pIMR174 GAT CAC ATC TGG TAT TGT C
poIMR0035 TGG GAA GCC CCT TTG CAC
poIMR0036 Fluorophore TGC AGC CCA ACC TCT GAT ACA

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR0035 0.50
poIMR0036 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

This is the only strain that uses this protocol.