Stock No: 006676
Protocol 31464: Probe Assay - Or8a1<tm26Mom> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr9:37729973-37730094 122bp AAGACCGCACTGGACAAAAC GAGTTGGGTGAGCTTGCAG

Mutant=  171 bp

Wild Type = 122 bp

Sequence

Wt Sequence: AAGACCGCACTGGACAAAACACTGAGGAGAAAAGTCTTTTGATGTaaatgctgatctttcttcatgtaaaagtaagtggttatgcacacctgtatgataacctctGCAAGCTCACCCAactc

 

Mutant Sequence: ATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGACTCTAGAATAACTTCGTATAATGTATGCTATACGAAGTTATTTAATTAAaaatgctgatctttcttcatgtaaaagtaagtggttatgcacacctgtatgataacctctgcaagctcacccaactc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
26816 GAT CAC TCT CGG CAT GGA C Transgene Forward A
36925 AAG ACC GCA CTG GAC AAA AC Wild type Forward A
36926 GAG TTG GGT GAG CTT GCA G Common A
36927 Fluorophore-1 CTG TAC AAG TAA AGC GGC CG Quencher-1 MUT Probe
36928 Fluorophore-2 CTG AGG AGA AAA GTC TTT TGA TGT AAA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
26816 0.40 uM
36925 0.40 uM
36926 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
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
006676 B6;129P2-Or8a1tm26Mom/MomJ
029558 STOCK Or8a1tm26Mom Tg(Or8a1,-MAPT/mCherry)4MGaFeins/J
029600 STOCK Tg(Or8a1-OR1A1,-MAPT/mCherry)D736Feins/J
3 strains use this protocol