Protocol 37923: Probe Assay - Fahd1<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

>chr17:24850316-24850451 136bp CACGCCCTCAATCCATCAG GCTAGGGCAGCTAGGGAACA

Mut= 106 bp

Wt= 136bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

ACTTACCCAGACCACGGCGCGCGCAGGTGGCTCCCAGCGCTGACAGGCTCCGGAGACACAGCGACCCGCGACCCAGCACCATGACCCACGGGGTGCACAGCCGGGTCGCGGAGTCCGCAACACCAGCGTCGTGGGCATCAACACTGGCGTTGGCGACTGCAACCAATCAGTGTCGTCCTCCTACTGCTCCCAACCAACCAACTCCTGCTCTTTACCAAAATGCCACGCCCTCAATCCATCAGTGACCGCCcatagagttcacagccaaccagtgcctgtctcgcaccgccgcgccgcccctagccaatcagtagtagctcctgccccccccccccaaaatgttccctagctgccctagcagccttggcctgtagactcggtctgcaagtgggtgttggaggaggctgagcgtgagcctcctcgtctctctggggcagtcgcctgcacgcagagacctttcgctgacctcagcgtcccgctgctgcgcaaggaagggcggggccgctccgacctggacagcgtccgaagccagtcctctggaggccgccgtagtccgggggagtcggtggtcacatgacccaaagctgtactatggcttccaccaaaccgctgtctcgtttctgggagtggggcaagaatatcgtctgcgtggggaggaactatgcagatcacgtcaaagagatgcgcagcaccgtgctgagtgagcctgtgcttttcctgaagccgtccaccgcgtacgctcccgagggctcaccggtgttaatgcccgcttactgccgcaacctccaccacgaggtggagttgggagtgcttctgggcaagcgtggtgaagcgatcccggaggctgcagccatggactacgtggccggctatgccctgtgcctggacatgactgccagagatgtgcaggaggagtgcaagaagaaggggctgccgtggaccctggctaagagctttacgtcctcctgcccggtcagtgccttcgtgcccaaggagaagattcctgaccctcatgccctaagactgtggctcaaggtcaacggagagctcaggcaggagggcaaaacatcatctatgatcttttccatcccctacatcatcagctatgtttctaagataataaccttggaagaaggagatcttatcttgaccgggactccaaagggagttgggccaattaaagaaaacgatgagatcgaggccggcatagatggggtggttagtatgaggttcaaggtgaaaagatcagaatactgagagtagagtgccaaaggggaagggagacagaagcaagggaaataaatgacactaataatgaaaatctaaaaattatgctagacatgtcaaaaaagatgaatccttaaaaaataacgtgatctaaaagacctgggactgaaatagaaacaggaacaacgaagctaaaggatatagaatgcttcccaagagggcaatgttaaactaaactttgagaagttgtagttttcttttctaaaactgaactgaatagacttttcaataaatcattctgaagtctatacctccagacttaaaagtgcaaaggaagtaactcattaatgctgttggcctttaggaaagctgtttgtacagagcactccagttgatttttacttgggaaagtcctccacctcaaagccagataagaattttgttaagtgagctcttgctgcagccctgccttgtttactttgggtgtggcttctgagtttggaagataattttgaaattgttttccaaataaagaccatttctgtgtgactgaaaagagcactttcttttcccagggtggggagcacttcttcctctgggacttaggaccttggcctttggtttcctgagcactgtgcctctttaacttattaaccacataaaccaatacttccacctcaaagacacaggccctagggaactgacaagcctggagatgctgtgtgtgttcatgcttgcacccctccctgataaaatgcctgtcaggatgcacagcacagttgtcagggcctagtggagtcttgtagaattagatagtggcttttgctgagagggtacataggagcctgctggtccctttaagaaggcatgttacatcttgttagtagtttatcattactgtaaggcaatatgccaaccatagttcattccatatttctgtgtattctgggtttctTGTTTGTTTTTTTTGTTTGTTTTGCAAAGCTATACCACTGGCATTATTATTATTTCTGATGACAATGCAGACTTAGAAGATAGAACTGAAGTAGAGCCAAGTTGTAATGAAATTGTCACAAACTCAGAACCTGAAGTTTTAACTACAGCATAGGTAGTGTCATATAAGTAGATATATAAGAAAATTTAAGCATTTAGGTATTCTGTGAGCCCCATGCTTAATTATTTATTATGAGGTCCCATAGTGCTAT

This mutation is a 1932 bp deletion beginning at Chromosome 17 position 24,848,493 bp and ending after 24,850,424 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50866 GCT AGG GCA GCT AGG GAA CA Wild type Reverse A
52524 CAC GCC CTC AAT CCA TCA G Common A
52525 CTT CTA AGT CTG CAT TGT CAT CAG Mutant Reverse A
52526 Fluorophore-1 ACC AGT GCC TGT CTC GCA C Quencher-1 WT Probe
52527 Fluorophore-2 TGC AAA GCT ATA CCA CTG GCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50866 0.40 uM
52524 0.40 uM
52525 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.