Protocol 41222: Probe Assay - Tmem229b<em1(IMPC)J>Alternate 1
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

Mut = 111 bp

Wt = 106 bp

Fam = Mut

Hex = Wt

>chr12:78961590+78961695 106bp ACTGCAGGAGTTAGGGAGCA AACCAGGATGGCTGGTGGT

Sequence

MUT Sequence (junction in uppercase):

acctccagcctgggactgcaggagttagggagcattggagtggacacaggtgACtaccttctgtatcacctcaactccctgttagcactggctttcttctgagaaactctacgcaaatactatgtgaaaatgtctactgaa

This mutation is a 3696 bp deletion beginning at Chromosome 12 position 79,008,403 bp and ending after 79,012,098 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
57776 ACT GCA GGA GTT AGG GAG CA Common A
57777 AAC CAG GAT GGC TGG TGG T Wild type Reverse A
57779 Fluorophore-1 TGG CAG CGC CAC ACC Quencher-1 WT Probe
58735 ACA TAG TAT TTG CGT AGA GTT TCT CAG Mutant Reverse A
58736 Fluorophore-2 CTC AAC TCC CTG TTA GCA CTG GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
57776 0.40 uM
57777 0.40 uM
58735 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.