Stock No: 000504
Protocol 2412: Restriction Enzyme Digest Assay - Cacnb4<lh>
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 = 199 bp with no Hph1 site
Wild type Cut = 160 bp and 35 bp
Wild type Uncut = 195 bp
Separated by gel electrophoresis on a 3% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR9387 CAA GAC TTT ATT TCC AAA CCA GTG Common
oIMR9388 CGC CTT GGA TTC TCA GTT TC Forward

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.00 mM
dNTP 0.20 mM
oIMR9387 1.00 uM
oIMR9388 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 58.0 --
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
Hph I

Strains Using This Protocol

This is the only strain that uses this protocol.