Stock No: 004753
Protocol 961: Restriction Enzyme Digest Assay - Tg(Il1rn)1Dih
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 = ~84 bp and 70 bp
Heterozygote = 70 bp, ~84 bp and 163 bp
Wild type = 163 bp
Separated by gel electrophoresis on a 3.0% agarose gel.**

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR3935 ACA CAC AGA CGC TCC CCT AC Common
oIMR3936 TCA TCT CCA GAC TTG GCA CA Common

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.00 mM
dNTP 0.20 mM
oIMR3935 1.00 uM
oIMR3936 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 63.0 --
4 72.0 -- repeat steps 2-4 for 40 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
Kpn I

Strains Using This Protocol

This is the only strain that uses this protocol.