Stock No: 006564
Protocol 14216: Pyrosequencing Assay - Gusb<mps>
Version 5.0

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 = -/-
Heterozygote = G/-
Wild type = G/G

Sequence

exon10--GCCCCGTACGTGGATGTTATCTGTGTAAACAGCTACTTTTCTTGGTATCATGAC
TATGGGCATTTGGAGGTGATTCAGCCACAGCTGAATAGCCAGTTTGAGAACTGGTATA
AGACGCATCAGAAGCCGATTATCCAGAGCGAGTATGGAGCAGACGCAATCCCAGGGATCCACGAG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
p116 AAC ATC CAC GTA CGG
poIMR0114 Fluorophore GTG TGA GGG GTA AAT TCA CTC GAC
poIMR0115 CAA ACT GGC TAT TCA GCT GTG G

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR0114 0.50
poIMR0115 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
006564 B6(C)-KitW-41J Gusbmps/BrkJ
006559 B6.C-H2-Kbm1/ByBir-Gusbmps/BrkJ
000256 B6.C-H2-Kbm1/ByBir-Gusbmps/J
006562 B6.CBy(Cg)-Gusbmps Gpi1a-m1J/BrkJ
002086 B6.Cg-Gusbmps Tg(Gussx)1Wat/J
006407 B6.Cg-Gusbmps/BrkJ
005053 NOD.Cg-Prkdcscid Gusbmps/SndsJ
001880 STOCK Gusbmps Tg(GUSB)4Sly/BirJ
8 strains use this protocol