Protocol 45413: Probe Assay - Sun3<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

>chr11:9028106-9028245 140bp GGGAGCTGTCCACACTGTTA GTTTGACATTTCCTGTAAAGATTGT

Mutant=  114 bp

Wild Type = 140 bp

Sequence

Wt Sequence: gggagctgtccacactgttaaaattctgcagattagggaattcaggtccatgtccatgaaaCagaaggtaaatgttcaagcagttggcaaagattaataacaaaaaactatttttacaatctttacagGAAATGTCAAAC

Mut Sequence: gggagctgtccacactgttaaaattctgcagattagggaattcaggtccatgtccatgaaaCCgtaggagacatacaccactgtatattattatgaaccacaatgacactcctt

 A 409 bp deletion beginning at Chromosome 11 position 9,027,775 bp and ending after 9,028,183 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65546 GGG AGC TGT CCA CAC TGT TA Common A
65547 AAG GAG TGT CAT TGT GGT TCA T Mutant Reverse A
65548 GTT TGA CAT TTC CTG TAA AGA TTG T Wild type Reverse A
65549 Fluorophore-1 AGG TAA ATG TTC AAG CAG TTG GC Quencher-1 WT Probe
65550 Fluorophore-2 CCA TGA AAC CGT AGG AGA CAT ACA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65546 0.40 uM
65547 0.40 uM
65548 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.