Stock No: 000632
Protocol 14298: Pyrosequencing Assay - Lep<ob>
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 = C/T
Wild type = C/C

Sequence

GCTCCGTGGCTCCCAGTCAGCTGATGACAGGAAGTAAGGGCCTGGACCAGGAAGGTG
AGAAGGAAGGAGGTAGCCCAGGTTCACAGATGTAATGAAGGGCTCTGGAGACCGATC
TCCCTGCCACTTGCTAAAGCACCTCTTGTTCTTCTTCCTCCTGCATAGCAGTCGGTATCC
GCCAAGCAGAGGGTCACTGGCTTGGACTTCATTCCTGGGCTTCACCCCATTCTGAGTTT
GTCCAAGATGGACCAGACTCTGGCAGTCTATCAACAGGTCCTCACCAGCCTGCCTTCCC
AAAATGTGCTGCAGATAGCCAATGACCTGGAGAATCTC(c/t)GAGACCTCCTCCATCTGCT
GGCCTTCTCCAAGAGCTGCTCCCTGCCTCAGACCAGTGGCCTGCAGAAGCCAGAGAGC
CTGGATGGCGTCCTGGAAGCCTCACTCTACTCCACAGAGGTGGTGGCTTTGAGCAGGCT
GCAGGGCTCTCTGCAGGACATTCTTCAACAGTTGGATGTTAGCCCTGAATGCTGAAGTTT
CAAAGGCCACCAGGCTCCCAAGAATCATGTAGAGGGAAGAAACCTTGGCTTCCAGGGG
TCTTCAGGAGAAGAGAGCCATGTGCACACATCCATCATTCATTTCTCTCCCTCCTGTAGA
CCACCCATCCAAAGGCATGACTCCACAATGCTTGACTCAAGTTATCCACACAACTTCAT
GAGCACAAGGAGGGGC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
poIMR0214 GTA TCC GCC AAG CAG AGG GT
poIMR0215 Fluorophore GGC CAC TGG TCT GAG GCA
poIMR0216 CCA ATG ACC TGG AGA AT

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR0214 0.50
poIMR0215 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
006906 B6.Cg-Lepob Ldlrtm1Her/J
000632 B6.Cg-Lepob/J
000696 BKS.Cg-Lepob/J
004824 BTBR.Cg-Lepob/WiscJ
4 strains use this protocol