Protocol 36281: Probe Assay - Ino80c<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

Mutant=  116 bp

Wild Type = 126 bp

Sequence

Wt Sequence (deletions in lower case):

AGCTCCGGCCTGGGTTTTCTGTCCAAATCAGACCCTTGATGTCCAGACTCTTAAGGCTAGCCTTTGTATTTCAAGACATAGTTGACAAGATCATCACGATATAAGGACTTTATATCATGTGGTGTCATGGAATAATTTCCATTGAAAAGTTGTGTGTTGTGTTGCTGTGGACCCTGACCCTCCattttctgtaaggcgggcatagttgcacctccttcgtatcagttgtaaatgagacagctggagtaggacagatgcctgagcttgacacagagaccagggcatatggcaactgggaggcctgctttgtgtgtgcagtggcctctgttcacacagagtttcccctggcctccagggcaggagctctacctgctggagccctgtgcatgtctgtctgctggagagcagaagtggggaacccatttggcttcaaagattttttttcccctgttttaagggtgtcagcattgaagccatgaatgagagtaaaatggcgtcctcggagttaagctcggggcctgtggaaaaagctgccaagccgttgccatttaaggatcccaactttgtggtaagcatatgaggtggtatatcaatacagggtgctgtaccttagcttctgatctagctgggcctgaggtggctctggagtaccctcttcctcagtcccatttgccaccttctcctgtgggctgttggagacggtagtcgggggttgggagtcgggggttggggggagagagtggcagttggcggaaaagggatttgggactggacaggaaaggccctttgtgatctcccagtgtagcagaactttgaggaggcatcttttccatgattgtctccctgtgcttttttactggctttaaataattttaaaagatgcttgaaatatacaactaaaataggagtagtcctcataaccctttatagccctttgattcatgaaggttatattaagtagccagttacaaattataactttaaaggtttattgtttttattaatgagttttggcaatgtactgtagccagggtctgcaaatatgttagtagtcttttccacttcatgcttttaatctttatagcacagaaagaaatttccagaaggcttgccacaccatgattgaagcttatttgagctttccaggaccactggtgattatgaggctcagtgacacacccttcgagggggtttagagatgtagtctcgagattaaatccaactgtgtggtaaacttattttattttattttttttattttttatttttttttggtttttcgagacagggtttctctgtatagccctggctgtcctggaactcactttgtagaccaggctggcctcgaactcagaaatccacctgcctctgcctcccaagtgctgggattaaaggcgtgcgccaccacgcccggccaacttattttaaaggaatgagatcataagtgaattcaactagattggatcacataaagaatttttcctgtgtgcatgctgaaaaatagcatttacttctgtcccatcaccagcaaacttgtgggtttgaagagcccccaaactaggtcctttttgtttgcttgtttattttcatcttttgttttttgttttttgataaaggtcacctgtagcccaggctggcctcaaactccctactcttctgcttctacctcctgagtgctggcattgtagtgacatccaccacgcatggctaaaaactatgttctttcaaacaaagatttaagtgtaggtgaggtacagatagttactgatggaccgagaggtgcctgacaaccctaaagggcaggggcagggcgtcacgcttctccaggcgcgtcttccactgacttctcttcaaaccttcctttaattcaagcactctggccatggcggcgctgtagctggcaagaagaacagaacctggaagaacctcaaacagatcctcgctgctgaaagggcgttgccatggcaactgaacgatcctaactgtgagctgtctagagccttccatgtgtttgtaatgcagacaggaacttccttacaacaagcctacgttatttgctcgtgtttgtgttacttcagggaaatattcttagcctgagacccgcacagtggcacccatccagggatagtctgcatgtattccctttccactgagttggtgttgaggctgtttgtttgtcacggctgtgtgggttttagtgtcattatttaaaatttcagtctggagaaataggtcccgccgagataatgttgatgcttctattaagcttcccattttaaagtctactctttaagtttgttttcacaaacaacgtagcccacctaatgtgagaactggaaagtctccagcgtgcaggtaccagccactctctcactctctcctcccactgtagcatgtcctggcctgggcagaatccgttggtctactcaagtgcattggaaactttaggttccaagacctctccacctcctgccccgctgttcgtctttcatgggggtgactcagagggtgacaagttagtcatctgggtgtggactgcttgagtctgggtgtgtggatccagctgatggctgtgtggcctttcttcattgcctgttccactcagctggagcctgtgaagtagcctgcgggtagatgaacctggccgttccactggaggaacgtgtctctggtagcaaggcaggaccccagctgaggcccacacttagacatgctgggctggcttttgagccgtctctttttaagtgtgtaatcacaaaggaatatttcaggactgctgtcatgtgttagcttagagtaaaggagggggagtcgggatagttagaaggttatttggtgatcattttcttttctttcttttagactttagtattgatgctcctccatccttcaagccagctaagaagtattctgatatttcaggccttcttgtgagtataattacatctgtctatattagcactaaatagatttaagttatgtctctctagACAGATGGCTCTGCCTGGAGTCCATGCTGTGACCTGAGTGTCTGTGTGGGTAACCTGGAGGGGCCATTGCTCCAGTAAGCAAGACACAGAATTTTTACTTTTTTCCCTGTGATAGTTAACTGTATGCGACAACATGAATGAACCACCTTTCTGTTTTACTCTTGAGGTTTTAAAATACTCATATACAATTGGTGGTAGTTTAAAAACAAAT

This mutation is a 2807 bp deletion beginning at Chromosome 18 position 24,111,655 bp and ending after 24,114,461 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48303 CCT CCC ACT GTA GCA TGT CC Wild type Forward A
48304 CTG AGT CAC CCC CAT GAA AG Wild type Reverse A
48305 TGT GTG TTG TGT TGC TGT GG Mutant Forward A
48306 CTT GCT TAC TGG AGC AAT GG Mutant Reverse A
48307 Fluorophore-1 CAA GTG CAT TGG AAA CTT TAG GTT Quencher-1 WT Probe
48308 Fluorophore-2 TGA CCC TCC ACA GAT GGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48303 0.40 uM
48304 0.40 uM
48305 0.40 uM
48306 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.