Protocol 32348: Probe Assay - Atp9b<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

>chr18:80912681-80912768 88bp TGATGTCCCCAGCGTTTG AAATGGGGTGGGATGAAACT

Mut= 97 bp

Wt= 88 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

GGGTGTGGGAGGAGGGATTTCTAAGGCTGGACCTGGGAGGAAGAGGACTGTGGGGCTGTTGTGATGGAGATGTAAAGTGAATAAAAAATAAATTATTGGGAAAAGAAGAATGGTGACTTTGTAATAGAATGTTCAAAGGCATAGGGTAATGATAGGTTAGCATTAGACAGAAAGATTAATGTCcaaggaatatagacaatcaatgttgattgctaactatgtagacttaggttagaccttttaaaaagcctatacaacctatacaatatatgtttatcttttgttttatgcagttgctgtgattggctaataaatgtttgtcaaaggaagaaagagctaaaagctcgaacagtatggcttggatgtccagagaagtgtgaagagaaacatcctaggaattctatcaaaaatcagaaatacaatgtatttacctttatacctggggtaagatgatcgcatcttcaatgtcagttgatgtccccagcgtttgccttctgatgacacatgtagaaactctgcttgttgataacactgtttggtagtttcatcccaccccatttcaaatactttatattttcttggttctgtctacttaagaaaatgcatacttttattattgctttcagttcatgataaacattcatgttataagaacataaaagtatggaaatagtaaagaacaggtaaacatattttagttctggggacatttgatatttgtttggagggaaaattagaataaatgttgaagcctgccaaggaatttcacaaaggaagtatacgatttgttggtcttcatgtgggctctctaacaagtggattgcatggctgggcctcagtaggagaggctatgcctactcctgctgggactagatgtcccagggtgggatggtacctaaggggggtggggtgggggcggtgctcccccttCTGTGAGAAGGGGAGGGGGGCGTGGGAGGAGGGATTTCTAAGGCTGGAACAAGATATATCTCTGTATTTATTTATAGACACCTTTTTATATCAAGCTGTTAATCTCATGCCTTGTCACATTGTTAACTCATTAGTTACTTGGAATACTCAAGTTTAAATATATTAATTAATTATTGTTTTTACTACTTTAAATTATATTATAAAACTAGGTTTATCATTATTTGGAATATTA

This mutation is a 748 bp deletion beginning at Chromosome 18 position 80,912,310 bp and ending after 80,913,057 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38886 TGA TGT CCC CAG CGT TTG Wild type Forward A
38887 AAA TGG GGT GGG ATG AAA CT Wild type Reverse A
38888 GGC ATA GGG TAA TGA TAG GTT AGC A Mutant Forward A
38889 TTG TTC CAG CCT TAG AAA TCC Mutant Reverse A
38890 Fluorophore-1 TGC TTG TTG ATA ACA CTG TTT GG Quencher-1 WT Probe
38891 Fluorophore-2 AAT GTC TGT GAG AAG GGG AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38886 0.40 uM
38887 0.40 uM
38888 0.40 uM
38889 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.