Protocol 35161: Probe Assay - Dupd1<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

>chr14:21686322-21686410 89bp CTCATATTCCCTCCCCAGGA ACCAGGTTTGAGAATCACTTTC

Mut= 95 bp

Wt= 89 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAAACCCATTAATGTTTGGAAGCTACAGAACAGGGGCCTCTTTTAAAGGTTCTCCCAGGGGCTCTGTGAAGCAGGCTCAGAGGAACAGATTGTGTTTCAGTTGCCTTTGGGGAGATCTGACAGGTCCACCTCCAGGACCTGCAGCGTAAGGTGAATGATCcaaagggaaagggcaaatgcccagactgcgcctacccagggctgggcagatggtagctttcttatagtcatgaaaccaaacggtgcccagtggcctggggctcgggaagagcgctgctttgggtctttgcctgtaatctccctgcgattactgggtccagcgttgaaagccgtgcaccacccaaagcgcggcgtccaccaaccgaccgacccttctccctgcgcccagggccacggcgctggaccgctatgggctgcagaaggcgggtttcacgcacgtgctgaacgcggcgcacggccgctggaatgtggacaccgggccggactactaccgagacatggccatcgagtaccacggcgtcgaggccgacgacgtgcccaccttcgacctcagcatctttttctactccgctgcggccttcatcgactcggcactccgagatgaccacagtaagtgaccctctccaactctgccctgacaaggccccgcccccaggctgaaaacccacccatcaggtcccgctcccagacataaaacacgccccccctccccgccagccgaggcaccacccctcccgcccacagtgtgctttccccctgctaggtcatgccccaaggcatgctatcctctcccctctggggagaggtcctgccctcatattccctccccaggatcttccaacccggccctgccccagtggatcAGAAGCAGGATCTACAAGAAAGTGATTCTCAAACCTGGTGTACCGGGGCTGAGGATTGAAGGCAGGGATTGGCTGAGCTAACTGCCTGGCCATGTGTGTTGCTGGTGGGGTTCGGGCTTGATATACAGTGTTGAGATAGGAAAGGGGGAG

This mutation is a 703 bp deletion beginning at Chromosome 14 position 21,686,361 bp and ending after 21,687,063 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45420 CTC ATA TTC CCT CCC CAG GA Wild type Forward A
45421 ACC AGG TTT GAG AAT CAC TTT C Common A
45422 CTT TGG GGA GAT CTG ACA GG Mutant Forward A
45423 Fluorophore-1 CTT CCA ACC CGG CCC Quencher-1 WT Probe
45424 Fluorophore-2 CCA GGA CCT GCA GCG TAA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45420 0.40 uM
45421 0.40 uM
45422 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.