Protocol 34917: Probe Assay - Abhd4<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

>chr14:54262556+54262662 107bp AGAGGAGAGAAAACTGAAGAGTGT TGTTCTGGAGACCTGGGAAG

Mutant=  95 bp

Wild Type = 107 bp

Sequence

Wt sequence with deletion in lower case

TTCCTGAGATTAAAATATTGACCAAATCAAACTCGGGGAGGAAATGTTTATTTGGCTTACAGGTTACAATCCATCATGAAGTTTAGAAGCCAAGGCAGTAACCTAGAGGCAAGACCTGAAGCAGAGACCATAGAGCAATGTGGCTTCCTGGCTTGCTTCCCATGGCTTCCTCAGCATGTCTTCTTTTATCATTATTATTATTATCATCATCATTATTACCCTTTTCTTCTGAGACAGGGTCTTTCTATGTGGCCCTCGTTGTCCTGGAACTCACTGTGGAGACTAGGAGGGCTTCCTAAAATGATCTCTTTAAGAGGAGAGAAAACTGAAGAGTGTATTCTAGGCAAAATGTCTGTgctctcaggaagcattcttgatgctttacccaggaattcttcccttcccaggtctccagaacaagttcctggcccgttatgtatccctcccaaaccagaacaagatctggacggtgactgtgagcccagaacaaaaggatcgcacccctctggtgatggtacatggctttgggggcggtgtgggcctctggatcctcaacatggattcactgagtgcccgccgcacacttcatacctttgatctgcttggttttgggcgaagctcaaggccaacattcccaagggacccagaaggagctgaagatgagtttgtggcctcaatagagacatggcgggagaccatgggaatccccaccatgatcctcctggggcacagtttgggaggattcctggccacttcttactctatcaagtaccctgaaaggtaacttaagctactcttctcactcatgagctgactctgccttcctacacgttccaatatttatcccagtaatgctctcattccccagcaactggaaaactatgctacctccaagcaaatacagtgtatcttctgtttaatgtagagttaaacatcttatcctggtggatccatggggctttcccctacgaccaactgaccctagtgagatccgtgcacctccaacctgggtcaaggctgtggcatctgtcctgggacgttccaatccactggctgttcttcgagtggctgggccttggggtgagtcacctcccgatgaaggactctgtctttaagccaaggccatccaattcaccaccactcaccttggcgcacctacctatctctgtcatcctcactggttcttaaacactaggcctgcacatagcattgttccttaaaaccattcatcagaaccatgtctctgtattggaggtttcaggtgattgagatatatatatatatatatatatattcccttcatctgtatcagcagatagaaccagagaaaggatatatatatatatatttccttcatctgtatcagcagatagaaccagagaaaggatatatatattcccttcatctgtatcagcagatagaaccagagaaaggaagggactctgatgtgccacttctctgctacatgagtgaattttgtgtgacaacctggtagagactcagggcagtagccaccattggatccctttccatggctcaccagcaccacagcaagccggactcagatcctcagcttctaacataacacaccatggcagacaggtcccactgagccatgccctgctccagagacacaccgcatctccctctctggatttcttagcactaagtagctgtaagaagattctgagatcgagacaggacacactatagtctgtagtgtcagaggctctgcatgctctcacctcacgctgtccacgtgcctccctttgaggagcttttgacactgccgacataagaaacaggtccatttgactgaaaaggtggtgacccctgttttgtacggatagcttatgtctacatagaacttttcagtagtcacttgttagactggctgaaacgtaatggcacctaacagagtggaaaatgagccctgacatataaggtagcacaggtgttctgggataaagggagcaagtaagagacatgggtgccaaagggaagtaggcagccagagggctgacctcagagtccagggccctctaacagactcactaccatccttcctggtctaaccatgctcccctcccctggtcttggatctggccaggtccatgcaaggtatcagctatggtgttgggtagctgcctggcagccatggaagactgtgctagagggacagcagggcctgcggctatattcatggtatctcccccagatatcgctcacagacaaaaggcctgccaggacagaggcagtgtccagtgggcccggtcctcacattctgcctgcatgcctgccgcttcttcacgcacagtggtgaaagccggggccttcagggctgttaaggtcacctttccaagtattctgttacagcccgaaagtgagagagtgcttgggaaatggtatggtttcattgctaccttcagaaaagtaaaaataaataaataaataatcgcaaggttacagggtggggttggggtggaggagggggaatgcataactctgacttgaaaatcctctcaaccacagaccctcccagaacgtgtgagcctgtaaggaaagtgctggggctagctgacatgaaaattaccaacttccgtggggatggtagactgtcactaaactcattggtgaccaaaaaatcaaactctgacatttgaagtcatctgttgcttcccaacaggctcctgcactctctgtcatgttgactgcataaaggaactaagttcgtcaagagatgaactgggagaaggttgtttaaacataagcgggcaggagttcctcttctctccctgccaccaccactcactctgaccgctccacggttttgtttgttttgttttttggtggtggtggtgggggatgttgttgttctttctgttctctggtaggggcttaaaatagggtctcacacatccccagaccgaccttgagcttgaaacagttctcctgcgtctgcctcctgagtgctggaatcccaggcacgtaccacttgcttgcccagtatcacgttgttcttcctaccccctcccagggcctgggctggtgcagagattccgtccagacttcaagcgcaagtttgcagacttctttgaggatgacaccatctcggaatacatctaccactgcaatgcacagaatcccaggtgagggcggtgcccaggaccagtgcggggtggggctggggaggggctgaaaagtaaggctttgagctactgcttctcttccacagaagcctgcctcgggggaccccaggcaccagcaggcatgtctccagagttccctaggggtggtcaccagagctctaggagacaatagacttaattaaatatgctgtccctctctagaggaagacctcatgGCTTCCCTAGCGATCAGTAGCTGACATGATGAGAAACCCTCTGGGAAACATTTAAAAGATAGTTTATAACTGAAAAACTGATAAAAAAATTTTTTTGGTGTTTTGGGTTTTATTTTATAGGCAAATGTGGGGGGGGTGTTTTTAAGGCAAATAGAAAAGTGAAGTCTTTGCCCATTCACAACCTTCAGTTTCTAGATTTTCTTATTTTTAGTCTAATGTTTCATGCCATATTGATCCAGAAAGACTTCTCTCATGTGTCCC

A 3008 bp deletion beginning at Chromosome 14 position 54,262,600 bp and ending after 54,265,607 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44793 AGA GGA GAG AAA ACT GAA GAG TGT Common A
44794 TGT TCT GGA GAC CTG GGA AG Wild type Reverse A
44795 ATG TTT CCC AGA GGG TTT CTC Mutant Reverse A
44796 Fluorophore-1 CTC AGG AAG CAT TCT TGA TGC Quencher-1 WT Probe
44797 Fluorophore-2 TGT CTG TGC TTC CCT AGC G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44793 0.40 uM
44794 0.40 uM
44795 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.