Protocol 37133: Probe Assay - Fsbp<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

>chr4:11579360+11579461 102bp GCATACATGCGTGGAAATGT GCTCCAGATAGGATTTAATGCAC

Mut = 91 bp

Wt = 102 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TTGTAAACAAATAACACAGAACAGTTCCTCTTTTGTTTTTATTAAAGTGACTGCGGTGCTGTTTCAGCCCCTCTGCCAGCATACATGCGTGGAAATGTGATATTCTCACTAAATTAGCTcattttaagctgtggttcctgcttatctccagacctttgtgcattaaatcctatctggagctgctccctgtatacacaagtgtgttttgcttgtgctctcctgccttctgtgagaagctagctttatgtaggtcaggcttggcttaagcggctttcctgaacctgcgtaagtcagcttaagctggaggaattttaataagccctcccctgtaggcgtgggcctaaatgaggctagcctagttaagcctgatctttctgtttgtgaaaagagctgagcagagctgaaggctgcatggtggtttcagaaacggcctacttaattccaaaagaacaatggtaggaaaggctagatcttccaattttaccttatctgaaaagcttgatttgctaaaccttgtgaagccatatgtgaaaattctcgaagagcacactaataaaaattcagtaatagtggaaaagaatagatgttgggatgtcatagcagttaactataatgcgattggagtagaccgccctcctcgcacagctcagggcctacgcagcctttacaaaaggctcaaagaatatgccaaacaggagctattgcagcaaaaggaggcccagtctgagtttaaaagcagtgtttcggagcccaccaagcaagtcatggaaatgattccccagattgccagcttcggcctcataagagacaggagccacatacagaggtaagccttattttattttgttgtgggaattatggcgcctcctatctcttgggctctggcttagctgtgctagacatgtgcatagcgagcgtccccacaaggggagccgtgaacagtgtctttgttttgctttcttaaaaaaaaaaaaaaaaagtaaagctctgaggagttctctgttaggacagcttgcagctgctttccctccatCCTGCCGATTCCACAAGTAAAAGTTAAGGCAGAATTGATGGGATGAAGCTGTGACATCACTAAGTGAACTTTAAAGCCTTCATAGCGTAGAGTGTATAAAGTAATGGGTTTTACTATGACATTTTTA

This mutation is a 914 bp deletion beginning at Chromosome 4 position 11,579,401 bp and ending after 11,580,314 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50616 GCA TAC ATG CGT GGA AAT GT Common A
50617 GCT CCA GAT AGG ATT TAA TGC AC Wild type Reverse A
50618 AGC TTC ATC CCA TCA ATT CTG Mutant Reverse A
50619 Fluorophore-1 AGC TGT GGT TCC TGC TTA TCT CC Quencher-1 WT Probe
50620 Fluorophore-2 TTA GCT CCT GCC GAT TCC AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50616 0.40 uM
50617 0.40 uM
50618 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.