Stock No: 032032
Protocol 37715: Probe Assay - Tnfrsf25<tm1.1Rsie>-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 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

>chr4:152117892+152117993 102bp TCCCTTCCACTCCCATATTTAG GCCAAGGGCTAGCTGCAAAG

Mut= 123 bp

Wt= 102 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence:

ttacgcggatacataaaagagcagatattatccctatcccttccactcccatatttagagtagacttaccgtttatttgcttacactgatagagctttgaaacttcagctaagactggctttgcagctagcccttggcaagttgaaagatttcaggtttaccctctgaacgtgggactg

Mutant Sequence:

AGCAGAACGGTAAAGGGTTACGCGGATACATAAAAGAGCAGATATTATCCCTATCCCTTCCACTCCCATATTTAGAGTAGACTTACCGTTTATTTGCTTACACTGATAGAGCTTTGAAACTTCAGGCCGGCCCAATTGTTCGAAGGCGCGCCATTTAAATGAAGTTCCTATACTATTTGAAGAATAGGAACTTCGGAATAGGAACTTCATTTAAATGGGTTACCATAACTTCGTATAGCATACATTATACGAAGTTATCCTAGGCTCGAGGCTAAGACTGGCTTTGCAGCTAGCCCTTGGCAAGTTGAAAGATTTCAGGTTTACCCTCTGAACGTGGGACTGCAATAAGGGGAAAGGATACCCAAGCCCCTGAGTATCCCCTGCTTCCCAGACACTGACTCTAGGTGCGAGGACCACTACCCA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51986 TCC CTT CCA CTC CCA TAT TTA G Wild type Forward A
51987 GCC AAG GGC TAG CTG CAA AG Common A
51988 GAA GAA TAG GAA CTT CGG AAT AGG Mutant Forward A
51989 Fluorophore-1 AGC TTT GAA ACT TCA GCT AAG ACT GG Quencher-1 WT Probe
51990 Fluorophore-2 ACG AAG TTA TCC TAG GCT CGA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51986 0.40 uM
51987 0.40 uM
51988 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.