Protocol 13638: Pyrosequencing Assay - Lepr<db>

FVB.BKS(D)-Lepr^db/ChuaJ

Stock No: 006654

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-Dock7^m Lepr^db Myo15^sh2-J/J
000699	B6.Cg-Dock7^m Lepr^db/+ +/J
005089	B6.Cg-Qki^qk-2J/GrsrJ
000642	BKS.Cg-Dock7^m +/+ Lepr^db/J
000700	BKS.Cg-Dock7^m Lepr^db/+ +/J
008340	BKS.Cg-Lepr^db Nos3^tm1Unc/RhrsJ
001192	BKS.Cg-mea^J Lepr^db +/+ + Dock7^m/J
000707	CBA.Cg-Dock7^m Lepr^db/+ +/J
006654	FVB.BKS(D)-Lepr^db/ChuaJ
9 strains use this protocol