Stock No: 002495
Protocol 342: Restriction Enzyme Digest Assay - Cola1<tm1Jae>
Version 1.1

Notes

Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands.
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 = 412 bp and 225 bp
Heterozygote = 667 bp, 412 bp and 225 bp
Wild type = 667 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
oIMR0810 TGG TTC TGG AAT GAG GAT GG Forward
oIMR0811 TGC CTC TGC TTC CTT AGT GC Reverse

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 0.89 X
MgCl2 1.33 mM
dNTP 0.18 mM
oIMR0810 1.19 uM
oIMR0811 1.19 uM
DNA Loading Dye 0.62 mM
Perfect Match (Strat 0.07 U/ul
Taq DNA Polymerase 0.02 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 --
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
Sph I

Strains Using This Protocol

This is the only strain that uses this protocol.