Protocol 45299: Probe Assay - Dus1l<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

>chr11:120795185-120795265 81bp CACCAGTGTCACCTTGATCCT CCACTGGCACAGACTTGTAAA

Mutant=  89 bp

Wild Type = 81 bp

Sequence

Wt Sequence: caccagtgtcaccttgatccttctccgtctcagtaccctccTgtcgagaaccaacagttctttacaagtctgtgccagtgg

Mut Seq:  caccagtgtcaccttgatccttctccgtctcagtaccctccTCatggggagagttaatccctgcctctgactgtg

 

A 434 bp deletion beginning at Chromosome 11 position 120,794,790 bp and ending after 120,795,223 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65223 CAC CAG TGT CAC CTT GAT CCT Common A
65224 CCA CTG GCA CAG ACT TGT AAA Wild type Reverse A
65225 CAC AGT CAG AGG CAG GGA TT Mutant Reverse A
65226 Fluorophore-1 TCT CAG TAC CCT CCT CAT GGG Quencher-1 MUT Probe
65227 Fluorophore-2 CTC CTG TCG AGA ACC AAC AGT T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65223 0.40 uM
65224 0.40 uM
65225 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.