Genotyping Protocols Database
Protocol 6115: Restriction Enzyme Digest Assay - Psen1<tm1Mpm>
Version 8.0

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 = 350 bp and 180 bp
Heterozygote = 180 bp, 350 bp and 530 bp
Wild type = 530 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
oIMR1586 AGG CAG GAA GAT CAC GTG TTC AAG TAC
oIMR1587 CAC ACG CAC ACT CTG ACA TGC ACA GGC

Reaction A

Component Final Concentration
ddH2O
KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. 1.00 X
dNTP 0.20 mM
oIMR1586 1.00 uM
oIMR1587 1.00 uM
DNA Loading Dye 0.69 mM
KAPATaq DNA Polymera No proofreading function. 1 error per 40000 nucleotides. Initial denature 2 min. 0.04 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 69.0 --
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
ddH2O 0.00
NEB Buffer 3 0.86 X
BSA 0.09
BsteII
PCR product

Cycling

Step Temp °C Time Note
1 60.0 -- USE MJ #7
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
004193 B6.129-Psen1tm1Mpm/J
004807 B6;129-Tg(APPSwe,tauP301L)1Lfa Psen1tm1Mpm/Mmjax
2 strains use this protocol