Protocol 35020: Probe Assay - Nphp3<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

>chr9:104005588+104005706 119bp AACTGCACAGATGTCACTGGT CAGAGTAAGCACTAATCCTGCAC

Mut= 122 bp

Wt= 119 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGAGGGAAACAAATGTGTCCCATGTCACACACACCCATGCTGTCAGACTTCCAGCAGGTTCCACTGCGAACAATAAAATGAGGGTCCGAAGAAGTATTTCTGTTACAGATGAGGAGACTGAGAACCAAATGTATTTTGACTTTATGAATAAATGTTTTTACCCCcccccctgaaaacttggtggcacaattctgtgattacggaaaaaacgtttcaaaattaaactctaccttttaaaaagatttttagggaaaccaaagaaaatgaaattcaggacttactgagggccaagcgagagttggagagcaaacttcagaggctgcaggctcaggggatccaagtgttcgaccctggggagtctgactcggatgacaactgcacagatgtcactggtgagctcatttcgtgtcccactttccacagagCTGTATGGATGCTCGAGATGGTCAGTCATCCTTTTGCTCTCTGTGTGCAGGATTAGTGCTTACTCTGCTCTTACAAATGGGAGACCTTCTGTGTCTCTCTCAGCTGTCAGGACATCATCTCCTCTAAGTGTATGATGTCAGGAGCAAC

This mutation is a 261 bp deletion beginning at Chromosome 9 position 104,005,379 bp and ending after 104,005,639 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45044 AAC TGC ACA GAT GTC ACT GGT Wild type Forward A
45045 CAG AGT AAG CAC TAA TCC TGC AC Common A
45046 TGA GGA GAC TGA GAA CCA AAT G Mutant Forward A
45047 Fluorophore-1 CGT GTC CCA CTT TCC ACA G Quencher-1 WT Probe
45048 Fluorophore-2 CCC CTG TAT GGA TGC TCG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45044 0.40 uM
45045 0.40 uM
45046 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.