Protocol 37192: Probe Assay - Atp6v1f<em1(IMPC)J>Alternate 1
Version 2.0

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

>chr6:29469774+29469932 159bp GGGTGGTGGTTTGTTTTGT CGTGGAGGGAACTGGTAATG

Mut = 129 bp

Wt = 159 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 TTCTCTTCTCTTCTCTTCCTCCTCCTCCTCTTCTTCCTCTTCCTCCTCCTCCTCCATTTTTTTTTTTGTGGTGGTGGGTGGTGGTTTGTTTTGTTAATAAGGCTCTTGTGTTTCTAGACTTTCATGCAGAcacagaggggctcctatttctctctcctctgcctctcctccacaggggcagcttaaagaacacactgacttgcaccctggtcttcattaccagttccctccacgagctgatgtgtggactccaaccctgagctgtgtggttctgagctcctatgtgtagttgcttcatccaagaaaagagaaggctgaaggccttatacctccggccactgtcaattcctcttcttctcccccaacaggcaatttctaaacagggatgacattggcatcattctcatcaaccagtacatcgcagagatggttcggcacgccctggatgcccaccagaggtccattccagctgtcctggagatcccgtccaaggagcatccctacgatgcagccaaagactccatcctgcgcagggccaagggcatgttcaccgctgaagacctgcgctagggctgctcagccctcagccctcctcatcaccaggcctctcctggtttgccatgctctatttaagtgtcaagccttggtttccagcccctcccttcctcttcactgagtggctcgtaagctgttggggttcggttgttcaggtctaggctgggtagagaaagaaagcccaactgtttctccactgcctcctccctgtgctgttaatagtatcgttgttgatattaaaggaattattctctccaaatgggtactggtatggatacgtgaaggttgcagcattgggggacattagacttgcaaatatggaaaagcaaggtgaagaataattggtggggggaataattcaaggactggtcacctggatcagagactgccttcagcgatggaggacaaagtttgagcccagtctttctacctttgctgcttcacctagggtcttcacggtgctgggaggggcctgcccAAGGGACAGAAGCAATTGCCTAGGATTGTCATTGTCATTCTAGATGAGTGTTCTCAATCTGTGGCTCTCCAAAACCACAGGGGTCCCCAAGACCATCAGAGATCACTGATATTTAAATT

This mutation is a 899 bp deletion beginning at Chromosome 6 position 29,469,831 bp and ending after 29,470,729 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49998 GGG TGG TGG TTT GTT TTG T Common A
49999 CGT GGA GGG AAC TGG TAA TG Wild type Reverse A
50000 TTT TGG AGA GCC ACA GAT TG Mutant Reverse A
50001 Fluorophore-1 TCT GCC TCT CCT CCA CAG GG Quencher-1 WT Probe
50769 Fluorophore-2 AGG GAC AGA AGC AAT TGC CTA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49998 0.40 uM
49999 0.40 uM
50000 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.