Stock No: 017681
Protocol 30655: Probe Assay - Idua<tm1.1Kmke> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Mutant=  95 bp

Wild Type = 113 bp

>chr5:109110273+109110385 113bp CATCCTGTGGGCTGAACA AGAGGGCTGAGGCTGTTG

Sequence

Wt Sequence:tccaaatttctacctcttcatctcctcccatcctgtgggctgaacagtataacagactcccaGTATACaaatggtgggagctagatattagggtaggaagccagatgctaggtatgagagagccaacagcctcagccctctgcttggcttatagATGGAGAACAACTCtggGCAGAGGTCTCAAAG

 

Mutant Sequence: catctcctcccaTCCTGTGGGCTGAACAGTATAACAGACTCCCAGTAgggctgcaggaattcgatatcaagcttatcgataccgtcgagggacctaATAACTTCGTATAGCATACATTATACGAAGTTatattaagggttattgaatatgatcggaattcctcgagcggccTACAAATGGTGGGAGCTAGATATTAGGGTAGGAAGCCAGATGCTAGGTATGAGAGAGCCAACAGCCTCAGCCCTCTGCTTGGCTTATAGATGGAGAACAACTCTAGGCAGAGGTCTCAAAGGCTGGGGCTGTGTTGGACAG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35127 CAT CCT GTG GGC TGA ACA Common A
35128 AGA GGG CTG AGG CTG TTG Wild type Reverse A
35129 Fluorophore-1 CCC AGT AGG GCT GCA GGA A Quencher-1 MUT Probe
35130 Fluorophore-2 ACT CCC AGT ATA CAA ATG GTG GGA Quencher-2 WT Probe
oIMR7882 CGA AGT TAT TAG GTC CCT CGA C Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35127 0.40 uM
35128 0.40 uM
oIMR7882 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 4.0 -- 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.