Stock No: 004608
Protocol 875: Restriction Enzyme Digest Assay - Htra2<mnd2>
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 = 54 bp, 62 bp, 141 bp and 244 bp
Wild type = 54 bp, 62 bp, 141bp, 71 bp and 173 bp
uncut PCR product = 501 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
oIMR4023 CAC ACT GAG GAT TCA AAC CAA GGT Common
oIMR4024 GAC CGA GGA CAT AAA CAG GGT GTA Common

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.50 mM
dNTP 0.20 mM
oIMR4023 1.00 uM
oIMR4024 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 70.0 --
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
Alu I U/ul

Strains Using This Protocol

This is the only strain that uses this protocol.