Protocol 37844: Probe Assay - Dmtf1l<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

>chrX:126815418-126815523 106bp GAGTATATTTGAAGAGGTTTCTGACTC TTCATTCTGAAGGCAAAGCA

Mut = 119 bp

Wt = 106 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

AAGTTGAGTATATTTGAAGAGGTTTCTGACTCAGTAACAGTTCAAATTATGAGCTCTGTCTTacttagtgaggactcagacaggaacctcatgctttgccttcagaatgaagctaatgaaatggaaaatcctactgaacctctgaataagaggccttgtttgtcctctgaagttgattctacttcgtatatgtcagctcttgaattcccactacaagaaatggagcaaagttttggtttggtcacaaccacagccacagaggtggctgaggaagagtttggtggggaaaatatgatgcaattcgaggttttaaaagatgacaaacaatatgaagttttccccttgcctaacaaggaagagtcagtagttagccaagcatggttcacaagtaaggaagctaaggatgctctaactcatcaaggacagaaatggaagcaggggatgtggtccaaggaagaaacagctattctgatgaacaacattgaaaggtatatgaaggaccatggagtagagaatcctgcagaaatcatttttaagatggcaaaaggtaaaagaaaagatttctacaggtctgtatcattgggtctgaatagacctttgttttcagtttatagaagagtggtccgaatgtatgatgacagaaaccatgtgggtaagtacagccctgaggaaatagagaagctcaaggagctctggcagaagcatggcaatgactggataacaataggggccgccatgggaagaagtccatcttcggtcaaagaccggtgccgactgatgaaggatacttgcaacacggggaaatggaccgaagaagaagaacagcttcttggcgatgtggttcatgaattgacatgcactgaggtggatgagaaagttacccatggtgtgtgttgggcaacagtggctcaacgagtgggtactcgctcagcaaagcagtgtcgtgctaaatggctcaactacttgaactggaaacagactggtggtattgagtggacaaggaaagatgaagtcactctcatccaaaggctagtagagcttgatgtatccgatgaaagtgaaattcgctgggatgaattagctaaaggatgggaaagtgttcgttcaccacaatggctgcgaaataaatggtggatcatcaaaaggcaaattacaaatcataaagattttgcattcccagtcctagtaagatgtcttcaacaagaatatgaaagccaaaatgccagcctcaggttttgggagaataaatcaggatctgaagtaccagatagcaaccctgatatcatttttcaacaagtcccctttggagtcacaagtatagaaaataataatgcatccatctgtactgaccccatgacaggattgcaaatttcattccagctcatccccagacccacaacagaattacctgatgctgttgttgactctgaaaccataaccctgcccaCTGGACCACAGTAGATATGTGAGAATCTCCCTTCTTTCCTTATGTGTTCCACTCCCTTACCTTAAACCTATTTCCTTCA

This mutation is a 1381 bp deletion beginning at Chromosome X position 126,814,082 bp and ending after 126,815,466 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52313 GAG TAT ATT TGA AGA GGT TTC TGA CTC Common A
52314 TTC ATT CTG AAG GCA AAG CA Wild type Reverse A
52315 AAG GGA GTG GAA CAC ATA AGG A Mutant Reverse A
52316 Fluorophore-1 CTT AGT GAG GAC TCA GAC AGG AAC C Quencher-1 WT Probe
52317 Fluorophore-2 CTT TGG TCT GGA CCA CAG TAG ATA TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52313 0.40 uM
52314 0.40 uM
52315 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.