Stock No: 002561
Protocol 2120: Restriction Enzyme Digest Assay - Nek8<jck>
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 = 308 bp
Heterozygote = 108 bp and 200 bp and 308 bp
Wild type = 108 bp and 200 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR4004 CTT CCC ACC TGT TGC TGT TT
oIMR4005 CAG TGG GCT TAC CAC CAT CT

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.00 mM
dNTP 0.20 mM
oIMR4004 1.00 uM
oIMR4005 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 67.0 --
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
BseYI

Strains Using This Protocol

This is the only strain that uses this protocol.