Stock No: 024744
Protocol 30870: Probe Assay - Ids<tm1Muen> 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=  134 bp

Wild Type = 152 bp

Sequence

Wt Sequence:aagaggggaggagctacttgcatAGTTGttcaattttaatttggcagtagggtattgaaagacctttcatattttattcaacaattttctttccctatttaagGTATATCTTCCAATCACAGTGATGATTATCCAtaTAGCTGGTCTTTTCCACCTTATCATCCATCCTCAGAGAAATATGAAAACACTAAGgtaggtctgtacaagg

 

Mutant Sequence: AACCCCAAAGAGGGGAGGAGCTACTTGCATAGTTGTTCAATTTTAATTTGGCAGTAGGGTATTGAAAGACCTTTCATATTTTATTCAACAATTTTCTTTCCCTATTTAAGGTATATCTTCCAATCACAGTGATGATTATCCATcgggatccgcggtctagacttctcttcccacacgCAGTCCTCTTTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35691 TGG CAG TAG GGT ATT GAA AGA Common A
35692 GAC TGC GTG TGG GAA GAG A Mutant Reverse A
35693 CCT TAG TGT TTT CAT ATT TCT CTG AGG Wild type Reverse A
35694 Fluorophore-1 ATC CAT ATA GCT GGT CTT TTC CAC CT Quencher-1 WT Probe
35695 Fluorophore-2 CGG GAT CCG CGG TCT AGA C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35691 0.40 uM
35692 0.40 uM
35693 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.