Stock No: 002662
Protocol 772: Restriction Enzyme Digest Assay - Fech<m1Pas>
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 = 80 bp
Heterozygote = 23 bp, 57 bp and 80 bp
Wild type = 57 bp and 23 bp
Separated digested products on a 5.0% agarose gel.**

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0565 CGA AAC CCT TGG AGA AGT TCA A Common
oIMR0566 TGT TTT GAA TGG GAA GTG TC Common

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 1.52 mM
dNTP 0.20 mM
oIMR0565 1.00 uM
oIMR0566 1.00 uM
DNA Loading Dye 0.69 mM
Taq DNA Polymerase 0.01 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 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
BSA mg/ml
BspHI U/ul

Strains Using This Protocol

This is the only strain that uses this protocol.