Protocol 36181: Probe Assay - Atp13a3<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

>chr16:30356912-30357023 112bp GTCTCGTCTCCATCGTACCC ACATGCTTCCTCTCAGAACACA

Mut= 109 bp

Wt= 112 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AAATAGGAGTCTAAAGGAAGTAAACCGCTAAAGTTTGATTACCACTATTGATATCTTTGATTTAAATAGTGTAGCTAGCTAGCAATTTTATTTTACTTCATTTATTTATTTTGGTTTTTGAGATAGGATTTCTCTGTGTATCCTTGGCTGTCCTAGAACTCAGTCTGTAGACACAGTTGGCCTCGAACTCAGATCCAGCTGCCTCTCTCTCCTGAGTGCTGAGATTAAATGTGTGAGCCGCCATGACCTAGCAGCTTTTTTATTTTTAAAAGTAGAGAATTTTATAGTCATGgaataagacttggaagcatgaatctcagaatggttgactttagccatatctaacataattcttcttctaaattattaatataattactgcatttttctgtcatagaaagttgatttatggagtaaatgaaattgctgtgaaagtgccttctgtttttaagcttctaattaaagaggtaagactttgtatataacccaacaaacagtagagtcatccatttggaaagagtcatgtttaagaagctgttcttagtttcctgctttgcttcatattttacttggaaggtaaaaattgaaaaattcttgtctagtagaaaaggtatgagtatgcattttttattatatagatttctttgtgtctcgtctccatcgtaccctggttaagtgtttgtgacttgctcccttgctcctttacttcacattgGCTAGTACTGGAAACTGAACATTTGTGTTCTGAGAGGAAGCATGTTAGTTCTTGCAGTGAGGATTCATTTATAATTGCCTGGGCTTTCTGAATAAAAGATTAGGAATGTGTATGGGAAGTTGGTGAGTAACTGCCCAGCAGAAAGACTTGCCAGTCTTTCTTTGGGTTTTGGCTATAGATAAAAAGTAGTATTGGGCGGATACTTTACTAGAAACAATGATGTTTTCCCACCACTTTCTTAGAGCAGCCCTTTGGTGCTGCCTGAGAGAATAGTTTCAGGTATGATTAAGACA

This mutation is a 423 bp deletion beginning at Chromosome 16 position 30,356,957 bp and ending after 30,357,379 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48044 GTC TCG TCT CCA TCG TAC CC Wild type Forward A
48045 ACA TGC TTC CTC TCA GAA CAC A Common A
48046 ATG TGT GAG CCG CCA TGA Mutant Forward A
48047 Fluorophore-1 ACT TGC TCC CTT GCT CCT TTA Quencher-1 WT Probe
48048 Fluorophore-2 AGA ATT TTA TAG TCA TGG CTA GTA CTG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48044 0.40 uM
48045 0.40 uM
48046 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.