Stock No: 012889
Protocol 5580: Restriction Enzyme Digest Assay - Cacna2d2<du>
Version 2.0

Notes

BspHI has 100% activity using both NEBUffers 2 and 4. Enzyme is supplied with NEBuffer 4. Cuts at 37C. 07/23/10 LMV
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 = 286 and 273 bp
Heterozygote = 286, 273 and 541 bp
Wild type = 541 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10504 ACC TAT CAG GCA AAA GGA CG Forward
10505 AGG GAT GGT GAT TGG TTG GA Reverse

Reaction A

Component Final Concentration
ddH2O
KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. 1.00
dNTP 0.20
10504 1.00
10505 1.00
DNA Loading Dye 0.69
KAPATaq DNA Polymera No proofreading function. 1 error per 40000 nucleotides. Initial denature 2 min. 0.03
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 --
4 72.0 --
5 -- Go to step 2-4, 35 times
6 72.0 --
7 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
ddH2O
NEB buffer 4 1.50
BspHI 1.25

Cycling

Step Temp °C Time Note
1 37.0 --
2 4.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
012889 B6N;TKDU-Myo5ad Cacna2d2du/J
000575 TKDU/DnJ
2 strains use this protocol