Protocol 43927: Probe Assay - Ankrd60<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

>chr2:173572614-173572742 129bp CCCCTTTGGCTCAAGATTTTA TCAGAATTCCTACGGGAAACA

Mut= 96 bp

Wt= 129 bp

Fam=Mut

Hex=Wt

The Mut Reverse primer (primer 64361) anneals over the nucleotide sequence containing mouse genomic variations rs223620354. 

Sequence

Wt Sequence (deletions in lower case):

TCTTCTTTTTGGGTCCAGCTTCCATTTGAACTAAGCCAAATTAATTTTCTAAAAAGCCTTAGCAGCATCGAATTTCAGGCTCCTGTGCGACCATCAAGGGAGAGACTTCCCCCACCCCTACCCCCACGCACTCCCGCCCCTTTGGCTCAAGATTTTAAAAGTATCTTTGCGTGATTCAGGGCCAGacgtggtctgaactgatgcagtgatttctctaggtcctaattttgttaaaaggaaaaactgtttcccgtaggaattctgatggatgatgctaccctgaagttctacgatgtcatccccggagcacttatttcattatgtatctggcattacgatggttggacggaactggttttggccgccgtggaaggggaccccagcaaggtggtttcgtgctttttttttcaatattagggggcgaggggaccccagtctttagtctttagtgtgaaacttttgaataaagtcctcagagtagggaaggaagaagagctttctgcatggagagtgtttccagacagtctgggcttgtgagtccctcgGGCTTGCTTATCAACAGTTCATAAACCCACTTACTCTACCCGCAGTGGGGCCCAGGTGCTGCATTGTCTGTCCACATGTTGGCCAAGTTGAAAATCGAATTTAATCCTAGTTAAAGAACATGGGGTGGTAGCCCAGTGGTCAGGGGCTTGTGTACTCAGGTTGGTCTACCTTGGG    

 

This mutation is a 360 bp deletion beginning at Chromosome 2 position 173,572,334 bp and ending after 173,572,693 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64359 CCC CTT TGG CTC AAG ATT TTA Common A
64360 TCA GAA TTC CTA CGG GAA ACA Wild type Reverse A
64361 CAC TGC GGG TAG AGT AAG TGG Mutant Reverse A
64362 Fluorophore-1 CGT GGT CTG AAC TGA TGC AGT Quencher-1 WT Probe
64363 Fluorophore-2 CCA GGG CTT GCT TAT CAA CAG TTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64359 0.40 uM
64360 0.40 uM
64361 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.