Stock No: 007559
Protocol 22792: Standard PCR Assay - Oprm1<tm1Kff>
Version 2.3

Notes

A ~2300 bp read-through band may be amplified from the mutant allele.
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 = 227 bp
Heterozygote = 227 bp and 470 bp
Wild type = 470 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

Sequence

Mutant sequence:

TCTTCACCCTCTGCACCATGAGTGTAGACCGCTACATTGCCGTCTGCCACCCGGTCAAGGCCCTGGATTTCCGTACCCCCCGAAATGCCAAAATTGTCAATGTCTGCAACTGGATCtaattctaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgctacacaagtggcctct

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR7415 GCC AGA GGC CAC TTG TGT AG Mutant A
oIMR8520 ATC TTC ACC CTC TGC ACC AT Common A
oIMR8521 TGC TGG GCT CAA GCT TTA AT Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR7415 0.50 uM
oIMR8520 0.50 uM
oIMR8521 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.