Protocol 37162: Probe Assay - Foxb2<em1(IMPC)J>Alternate 1
Version 2.0

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

>chr19:16873547-16873655 109bp GAAGAGCCTGGGAAGATGC TCTCAGCCGAATGCTGGA

Mut = 108 bp

Wt = 109 bp

Fam = Mut

Hex = Wt

 

Sequence

MUT Sequence (junction in uppercase):

acttaagcctggtatccggctttccgagaggcgttaaggcccactgtgggccggacggaagtggaagagcctgggaagatgccacgGTtaggggaccccgcgaccggtgagaaggcgcctgtcgagagactgagacaatggtcccagaccgagagggtgctccaggaaggtcagcaaactt

This mutation is a 1274 bp deletion beginning at Chromosome 19 position 16,872,358 bp and ending after 16,873,631 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50701 GAA GAG CCT GGG AAG ATG C Common A
50702 TCT CAG CCG AAT GCT GGA Wild type Reverse A
50703 ACC TTC CTG GAG CAC CCT CT Mutant Reverse A
50704 Fluorophore-1 TCC TAC AGC GAC CAA AAG CC Quencher-1 WT Probe
50705 Fluorophore-2 CGG TTA GGG GAC CCC G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50701 0.40 uM
50702 0.40 uM
50703 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.