Protocol 45711: Probe Assay - Rhobtb2<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

>chr14:69799840-69799955 116bp GCAGCAAGGAGTGTGCTCTT GGGAGGACAGGACTGGTACA

Mutant=  88 bp

Wild Type = 116 bp

Sequence

Wt Sequence: gcagcaaggagtgtgctcttttaaGgtctcccctgcttccgctcagcgtggagattgccagtgatttcatgtgggcagcaggttgtacaagacacttgtaccagtcctgtcctccc

Mut Sequence:  gcagcaaggagtgtgctcttttaaGGcaggtgggtagtcagggccctgtgactgcttggggccgattctccagcatatccactgtgtc

 

A 3821 bp deletion beginning at Chromosome 14 position 69,796,110 bp and ending after 69,799,930 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66275 GCA GCA AGG AGT GTG CTC TT Common A
66276 GAC ACA GTG GAT ATG CTG GAG A Mutant Reverse A
66277 GGG AGG ACA GGA CTG GTA CA Wild type Reverse A
66278 Fluorophore-1 AGG CAG GTG GGT AGT CAG G Quencher-1 MUT Probe
66279 Fluorophore-2 CAG CGT GGA GAT TGC CAG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66275 0.40 uM
66276 0.40 uM
66277 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.