Protocol 45656: Probe Assay - Cfap100<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

>chr6:90412353-90412458 106bp CACAGTTCCGGGTCCTCTAA CTTTCCACAGGAAGGCTGTC

Mutant=  86 bp

Wild Type = 106 bp

Sequence

Wt Sequence: cacagttccgggtcctctaaaatcaagatccaGcagtggtcatatgggttctctttgcttccagcagGATCTGGCAGTAAGAGCAAGACAGCCTTCCTGTGGAAAG

Mut Sequence:  cacagttccgggtcctctaaaatcaagatccaGGggagggtgaggagagggaagacagctgaggccattccacggagctacctcag

 

A 349 bp deletion beginning at Chromosome 6 position 90,412,077 bp and ending after 90,412,425 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66149 CAC AGT TCC GGG TCC TCT AA Common A
66150 CTT TCC ACA GGA AGG CTG TC Wild type Reverse A
66151 CTG AGG TAG CTC CGT GGA AT Mutant Reverse A
66152 Fluorophore-1 TGA GGA GAG GGA AGA CAG CT Quencher-1 MUT Probe
66153 Fluorophore-2 AGT GGT CAT ATG GGT TCT CTT TG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66149 0.40 uM
66150 0.40 uM
66151 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.