Protocol 35139: Probe Assay - Cox16<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

>chr12:81485273-81485363 91bp AGTTACAACTGATGAGCCACGA CAGATAAAGGGCGGGGTTG

Mut= 91 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGTAGGATAGAGGGTTTGCGGAGCAGAAACCGGGACGGGGGATAGCATTTGAATTGTAAATAAATAAAATAATAAAAAATTTTAAAAAGTTCAGTTACAACTGATGAGCCACGACTTAAAATGTGTTCATTAACCTGGTGctacttgtgcattagcttaaaaaccaaccccgccctttatctgagtttcttgagtcgcagaagtcaagatccagaatatgcgaattacagattttccaaatcgaaaaacccaattgcacccggcgtatcctgaggtcacggaaccagagctcagaagcctcgacttccgggggagggaggaagcagaagtgcgttaaggccaatcgtttccgtttgcctcggggaggcttatcaggcgtggacctggcacccaaagcctgcgctgagtctacaacttcgcctgagacgtgttgcgggccgcgctgggctcgctctggaagcggctgactaagcaggccgcaccttctgccatgattgcgcccgctgtgttgcgagctctgcgtaagaacaagactctacgctatggagttcccatgttggtgagcggctgtgacgggagcaggggctgcgagtccggaaggggcgggccctctttggctgagtggcgtattggtgctgtctttcggatactgctgactagggggtctgtgggagccaccggagctttgaggtttttgtttttgtttttaagttcacgctcaaaagtgtacctcgggtcccgccatcccccaccccccgtttcctttcccttcgtgaacgcgcgtgTTACTATCCCCGTTTTACAGTACCTTTCCTGGGTTAAATGTTGAGATTTTGCAAGAAGACTAATCAAATGTCTTCTTTAATAATTACATTCATTTGTAGTAATTATGTTCATAATTTGTTGTTTG

This mutation is a 645 bp deletion beginning at Chromosome 12 position 81,484,671 bp and ending after 81,485,315 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45359 AGT TAC AAC TGA TGA GCC ACG A Common A
45360 CAG ATA AAG GGC GGG GTT G Wild type Reverse A
45361 CAA CAT TTA ACC CAG GAA AGG Mutant Reverse A
45362 Fluorophore-1 CTA CTT GTG CAT TAG CTT AAA AAC Quencher-1 WT Probe
45363 Fluorophore-2 AAC CTG GTG TTA CTA TCC CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45359 0.40 uM
45360 0.40 uM
45361 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.