Protocol 43654: Probe Assay - Cops9<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

Mutant=  129 bp

Wild Type = 129 bp

  
>chr1:92642119-92642247 129bp CCCATCTTCTAGCCAATCCA ACACCGCACAGCACGAGTT

Sequence

Large deletion:  Mutant sequence with junction in uppercase

cccatcttctagccaatccattactgcctgcgaatcctaaacagtagcCTctttatcagagctgctgctagtacaagaccccaggtcagaggactccgggtgccccgggtggagactctgaaggaggtg

This mutation is a 5306 bp deletion beginning at Chromosome 1 position 92,636,893 bp and ending after 92,642,198 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
63604 CCC ATC TTC TAG CCA ATC CA Common A
63605 ACA CCG CAC AGC ACG AGT T Wild type Reverse A
63606 CAC CTC CTT CAG AGT CTC CA Mutant Reverse A
63607 Fluorophore-1 AAC AGT AGC CAT TCG GAT GGT T Quencher-1 WT Probe
63608 Fluorophore-2 CAG TAG CCT CTT TAT CAG AGC TGC TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
63604 0.40 uM
63605 0.40 uM
63606 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.