Protocol 37191: Probe Assay - Dzip1l<em1(IMPC)J>Alternate 1
Version 2.0

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

>chr9:99640090+99640200 111bp CCTCTGACTTGTGCCCTTCA GCCCCATTTATACAGTCAGC

Mut = 123 bp

Wt = 111 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 ATCAGGATTTCTGTTTTCTTTCCCTTCCCACTCCCACCGTTCTTTCTGTCTTTCTGTGGTGTTGGGAATTGAACCTAGGGTCTCACCCACTCCCGACCCTTCAAATTCTTTTTTATTTCTGAGGCAGCCCATGGACCCTGTGGAGGCAGAGTTTCACTACAGGATATTTccacgctgtctgggggatctaatccagcccttccaattgcttttcttctgtgtagtgccacttgtgtgacaagaccttcatgaatgccacctttctccggggccacatccaacgcagacatgcaggcatggcagatgtcggtgagtccaggcttcattggggaactgctgggcaaggatttggtgtgtgaggcccatgcacatagacctgctcttagagcctcttagcgattccacatggtgccctgtgcagtcttgcctgtgtaggggacaagaggcatcagacaggtaggtttagttttagttttccatgaacagtggccagtacttagcctctgacttgtgcccttcactgtgtgtggagcaggcagtgactcataagggcctttagcctccctgtaGCCTCATCTGTAGCAATCCGAGGCTGACTGTATAAATGGGGCAAATGAGCAGTCAGACCAGTTCTCTGGCTGTTCTGTGTACGGAGCTTATCACGACTTAATCACTTTATTTAATTGTACCATTTCAGATTCCAAAAGGAGTAACAA

This mutation is a 390 bp deletion beginning at Chromosome 9 position 99,639,764 bp and ending after 99,640,153 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49330 GCC CCA TTT ATA CAG TCA GC Common A
49331 CCT CTG ACT TGT GCC CTT CA Wild type Forward A
49332 ACT CCC GAC CCT TCA AAT TC Mutant Forward A
49333 Fluorophore-1 AGC AGG CAG TGA CTC ATA AGG G Quencher-1 WT Probe
50768 Fluorophore-2 AGC CCA TGG ACC CTG TGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49330 0.40 uM
49331 0.40 uM
49332 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.