Protocol 37959: Probe Assay - Atp6v0b<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

>chr4:117887454-117887547 94bp GGATCTGCAGTGATTGGTGA CGTTCTCCGTGACAGGTACT

Mut= 87 bp

Wt= 94 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

TATAAGATCACCTGGACATCGTGGGCGTTCGCGTCTGGCATTCCACCCTACCTCTGGGTTGGAAAAGACAACCTAGAATGACCTCCGATGAACAGCAGGCATTAGCTAGGCACCGCGAAATCCTGCTTCAAGCAGAAGGAACTAGGCAGGACTAGAACAGACCGGAAGGATCTGCAGTGATTGGTGAGTAAACTGGGAGTCcggtgggaagttagggaaccagcagcgcaggtggagagccagtacctgtcacggagaacgtccgacgaaactacaaccaccacagtgctccgcggcatgacgtctaccagggcccggcctcaggcagccagccactgtgcgcgggggtcacgtggggtcggcgcatcacgtgcgccggtcaggtggcgctgcggggcgggccgacagactgcggggcggacggtggacgcggtttagctcaggtcccaccgatccgacccctgccgtcgccgctgccatgacggggctggagttgctctacctcgggatctttgtggccttctgggcctgcatggtcgttgtgggtgagcggggcccaggcggcccaaggtggcctgctggttggggcggggggcaggtggcagccggagccggccccgcgcggtgccctcccgcgacggaggaggccggccctgggctgcgagagtcacaccggctattccagggtcgcgacgccaggttcctgacctcctctccgcgagccgcatatttgggcgccccgcttcccctttttcctccttgccctcccccacccattcacagcttgttgggtaaacagcgattgggccggcaggatctggcgcggcctggacgcagcgcccgttccctccggcccgtgaaggggcttggtgtcattaagacgggttacctagggagtggccgaaaaaaaaaaaaaaatggaggcctgggtggaggtgctgtttcgctccgggactcttcgcttgtctagatgtaggtggctgacagtttgacagttgggcagttttttttttttcctaggttggggactgtgtctcgaaagagcaaccctttaactcctttcctcattgcttcaggaatctgctacaccatctttgacctgggctttcgctttgatgtggcatggtaagggagggcatggagaggattctcctaggagcctcttctccctgctttggtctgagccccagcccagctcttcagacacacggacctttgggaaatagaaaatctagagatgcaacgtctgggactggtgggctggagctgggtggacactgagttttgtgtggcctccccactcctgccctttcctgtccatagctcaatagctttcttccctgtttccacaccaggttcctgacggaaacttcccccttcatgtggtcgaatctgggcattggcctagcaatttctctgtctgtggttggagcagcctggtgagtgttggagtggcgggacgagtgggaggtgccaagtcgtggcaggtggaatcatcactgggttcttaatcatcctggggacagcaactccttgggctcctttaatcagtttcttacccttcaccaggggaatctatataaccggctcatccattattgggggtggggtgaaagcccccagaatcaaaaccaagaacttggttaggtaagtgtaaaggcttctgcatgtcaaaactagctgcgatgggggtcgggtgacatgctggcttgtgacccgtctcatcgtagcgtgtatgtgagtcagggaaagttggctggggattttctgcagaaatggctgatttccttcttaaagagtttccctccccctcccccaccatttaccatcactgttgccagtattatcttctgtgaagcggtggccatctatggcatcatcatggcaattgtcatcagcaacatggctgaggtaagggaaaaccagcggggacgggtatccattgaaatgcctatgctttccctaaagatcggagggaagaaaaggtaggtgcattaaggaatgccttctgaggacttattctgcccctcacagcctttcagtgctacggagcccaaggccattggccatcgaaactaccatgcaggtaagtgggtaacatagagctcactcggcatggtgctctgggcctttaaccccagtcctgggacgggggtagcaccaagtgagttaagaggccagccagcactacataatgacatagtgagacagacaccctaccacacatacacgcctgggccttacacacttgcttctaccacacgtacacacctgggccttacacacttactcctgcttcctggctccatagctgagagtctcagctctgcagagttggggtgggactggccctgctagcccacactggaccattcctctcctgcttgcctccacctcacgagctacttcttatcctccaggttactccatgtttggggctggcctcacagtcggtctgtccaacctgttctgtggagtctgcgtgggcatcgtgggcagtggggccgccctggcggatgcacagaaccccagcctctttgtaaaaattctcatcgtggagatctttggcagtgccattggcctctttggggtcattgttgcaatccttcaggtgcgagtccccctagagaggaacagaagtgcctcccttttttccccttcccatctcttggtccccctttgtctcctgctctagatgcacgcatctttcctaagtctctccattagcatctctttcctcggctgcctctgaaaatgtctttgtggagtttctgtctctcagggtctgtgttctgtatgtatgacacacttgtcagtagactagcatgtctaaattcatattttccagtgtgtgagtttgtgacttgggtgcttgactgggctgactcttggcgacactaacactgtccatgggacttgtcttctctgttgtacagctgagctcctttgttactgtccatgtctgtttgtctgtcatagtgtgtctgtccgtctgtccacccactggcctgctggttctggtcttgtcactggtcactgtccctgcctgatttgtcctgtttttcctttgcagacctccagagtgaagatgggtgactagatgacctgtatgggtggggccgtgccctactgttatttattgctggttttcctgggacagctggagctgtgtccctcagcctttctggggcttggtgttcagggtctcccctgcactcacctcttgctgcctgttgactctggaggcattgcagtccagactgaaccccagtgtggggagtgggctgctgatggtgactgtgtacagctgtgcacctgtgcccccctcaacccccatccagcccatcttcctagtgtttgtgaaataaacttggtatttgtcccagtcagtgaggcttctgttggccccttgcgacacctttcacaaaggaaggggaggacctgttcactcactccaacctcctgtaactttctgtctacacatcatctctgctctggaccttgctggctcctgggtggcagatcagggtgctaggctggctgtaatgctggcacctcttcattgaagagtctgggctctgagatcctcctagccctatgtctttggacccctgtgcactagacccAAACTCACTACAGCTAATTCTCCTCTCTGTCGCTATACTCCTGAAACTCCCCGCACCTGGATTTTCCTGGAGAACAGTGTTTTACATTTGATAGTCCAGTATGAACACCTAACCCTTCCAGAAACCTCTTCTATGGTCTTAGTTCCTAGGGCCTTGTTCAGCTCCGCCTATTCCTATACAATTCTTGATTCCAACACCA

This mutation is a 3425 bp deletion beginning at Chromosome 4 position 117,884,089 bp and ending after 117,887,513 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52624 GGA TCT GCA GTG ATT GGT GA Common A
52625 CGT TCT CCG TGA CAG GTA CT Wild type Reverse A
52626 CGG GGA GTT TCA GGA GTA TAG Mutant Reverse A
52627 Fluorophore-1 AGC AGC GCA GGT GGA G Quencher-1 WT Probe
52628 Fluorophore-2 CTG GGA GTC AAA CTC ACT ACA GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52624 0.40 uM
52625 0.40 uM
52626 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.