Protocol 45307: Probe Assay - Fmo1<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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

>chr1:162850466-162850572 107bp CCCAGGACACAATCAGTAAATC AGAACCTTGGGGGTAGTTCA

Mutant=  85 bp

Wild Type = 107 bp

Sequence

Wt Sequence: cccaggacacaatcagtaaatcagaacttaattcttaTttgcatatgtgcattcaatagaaacaaaaaaaatagattatttgaaccttgaactacccccaaggttct

Mut Sequence: cccaggacacaatcagtaaatcagaacttaattcttaTAcctggttagtcccaggatgaactcttgtggattgtccgttaaacag

 

A 526 bp deletion beginning at Chromosome 1 position 162,850,009 bp and ending after 162,850,534 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65252 CCC AGG ACA CAA TCA GTA AAT C Common A
65253 AGA ACC TTG GGG GTA GTT CA Wild type Reverse A
65254 CTG TTT AAC GGA CAA TCC ACA A Mutant Reverse A
65255 Fluorophore-1 TGC ATA TGT GCA TTC AAT AGA AAC Quencher-1 WT Probe
65256 Fluorophore-2 TTC TTA TAC CTG GTT AGT CCC AGG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65252 0.40 uM
65253 0.40 uM
65254 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

This is the only strain that uses this protocol.