Stock No: 006407
Protocol 764: Restriction Enzyme Digest Assay - Gusb<mps>
Version 1.1

Notes

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 = 95 bp
Heterozygote = 18 bp, 77 bp and 95 bp
Wild type = 77 bp and 18 bp
Separate by gel electrophoresis on a 5.0% agarose gel.**

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR6214 CCT GTC TCA TTT GCA TGT G
oIMR6215 GAT AAC ATC CAC GTA CCG G

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.00 mM
dNTP 0.20 mM
oIMR6214 1.00 uM
oIMR6215 1.00 uM
DNA Loading Dye 0.69 mM
Taq DNA Polymerase 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 55.0 --
4 72.0 -- repeat steps 2-4 for 35 cycles
5 72.0 --
6 10.0 -- hold**
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Reaction B

Component Final Concentration
AB PCR BufferII X
ddH2O
BSA mg/ml
NciI

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