Protocol 31359: Probe Assay - Rpain<em1(IMPC)J> 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

>chr11:70972850+70972947 98bp TCATGAGACATGTGGCCAGT GGTTTAATCCCTGAGCAAGACA

Mutant=  94 bp

Wild Type = 98 bp

Sequence

Wt Sequence: tcatgagacatgtggccagtTAatacttcatgtggagtgggaaaacaccactttctctctgctctgtctctgtttatgtcttgctcagggattaaacc

 

Mutant Sequence: tcatgagacatgtggccagtTCTGCTCAGAGAGgcagagtcttccctacctatgagaccgaccTCAGgcctggctcaggattaggtttgctgtt

215 bp deletion beginning at Chromosome 11 positive strand position 70,972,871 bp and ending after 70,973,085 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36692 TCA TGA GAC ATG TGG CCA GT Common A
36693 GGT TTA ATC CCT GAG CAA GAC A Wild type Reverse A
36694 AAC AGC AAA CCT AAT CCT GAG C Mutant Reverse A
36695 Fluorophore-1 ATG TGG AGT GGG AAA ACA CC Quencher-1 WT Probe
36696 Fluorophore-2 AGT CTT CCC TAC CTA TGA GAC CG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36692 0.40 uM
36693 0.40 uM
36694 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.