Stock No: 000642
Protocol 13638: Pyrosequencing Assay - Lepr<db>
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 = G/T
Wild type = G/G

Sequence

TCTATTTTATTTTATTAAACAGAGAACGGACACTCTTTGAAGTCTCTCATGACCACTAC
AGATGAACCCAATCTACCAACTTCCCAACAGTCCATACAATATTAGAAGATGTTTACAT
TTTGATGGAGG(g/t)AAACAAACCTAAACTATGGTTTGAATGACTAAGAAATAACATTTG
ATGAGCTTATTAGAGAAGTGTATATTTTGTGGCCACAATGTAGGTTTGATGTAGTTCAGT
TTGGACATATGCTTGATTTTCAGGGCATCAAAAATTTAAAGTTGATATTCATGGACTCTG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
poIMR011 GAT GAA CCC AAT CTA CCA ACT TCC
poIMR012 Fluorophore TGG CCA CAA AAT ATA CAC TTC TCT
poIMR013 TTT ACA TTT TGA TGG AGG

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR011 0.50
poIMR012 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
002048 B6 x C57BLKS-Dock7m Leprdb Myo15sh2-J/J
000699 B6.Cg-Dock7m Leprdb/+ +/J
005089 B6.Cg-Qkqk-2J/GrsrJ
000642 BKS.Cg-Dock7m +/+ Leprdb/J
000700 BKS.Cg-Dock7m Leprdb/+ +/J
008340 BKS.Cg-Leprdb Nos3tm1Unc/RhrsJ
001192 BKS.Cg-meaJ Leprdb +/+ + Dock7m/J
000707 CBA.Cg-Dock7m Leprdb/+ +/J
006654 FVB.BKS(D)-Leprdb/ChuaJ
9 strains use this protocol