Protocol 32269: Probe Assay - Fyco1<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

>chr9:123840401-123840500 100bp TCTCCTTCCCCATTTGAGTG GCTTTGACTCTTAGTCTCACCACA

Mut= 96 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

CATACCAACTATTTGTTGACTTGCTGTATATACATAGTGCTAGGTTGGTGGTAGATACAGGGATGAATTTTTTAAAAAACAAAACAAAAAACCCCACCCGTGCCCAATCAGCAAGCTGTTTCAGTGTCTATGGGGACTTAGTACACTTGACTCTCCTACCTGaatagttgaatgtcctgtgtgtttccaaggttacacttactccttgtgcttcttggtgttatttctacttctttctcagacttacttttcagtacatctaagatgcgtgtcaattctagggctgttaaaatgtgaagtagacaaatgtgtctctctagttacgtgttcctatttgttgtcttaaagatgcagcgacagaactaagccatgaatttaaggaaggcggagagccaatcacagatgacagcaccagtttgcacaagttttcttacaaacttgagtatcttctgcaagtaagtggcccctgtcatgcactggttttgtcagagttgtgtctccttccccatttgagtgactgttagcagtttccagggtgTGGTTCGGAAAGCACAGTGAAGCAGTTAAGTCCTTGTGGTGAGACTAAGAGTCAAAGCAGTGCCAGGAGCTCAGTCCTTCCACGTGAGTCAGGGTGTTCTGGGAACTGAAAACATTCTTCTGAGACTTAAGCCATCGTAAGGCAAGTCACAGGCCTTTCCAGCATTACACCCTTTGAGAACTTGTCGA

 

This mutation is a 376 bp deletion beginning at Chromosome 9 negative strand position 123,840,459 bp and ending after 123,840,834 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38712 TCT CCT TCC CCA TTT GAG TG Wild type Forward A
38713 GCT TTG ACT CTT AGT CTC ACC ACA Common A
38714 TGT CTA TGG GGA CTT AGT ACA CTT G Mutant Forward A
38715 Fluorophore-1 ACT GTT AGC AGT TTC CAG GGT G Quencher-1 WT Probe
38716 Fluorophore-2 TCT CCT ACC TGT GGT TCG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38712 0.40 uM
38713 0.40 uM
38714 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.