Protocol 38254: Probe Assay - Wdr53<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

>chr16:32256464+32256577 114bp GCTGTGGAGTCTTCAAAAAGC GCTAGCGCAGGGTTTAGGA

Mut = 109 bp

Wt = 114 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GACTGTAATTTCACCTTGTTTAAAGGTAATGCTGTGGAGTCTTCAAAAAGCCCGTCCTgtttggattacaaatttacaggaggatgaaacagaggaaacggaaggcccccagacccctggtcggctcctaaaccctgcgctagcccactctgtctctgtagcgtcgtgcggcaatatttttagctgtggtgcagaggatggtaaggtgcgcatcttcagggtgatgggagtcaaatgtgagcgagaactgggatttaagggtcacactttgggggtatcccaggtctgctttctgcccgagtccagtctgttgcttactggagggaatgatgggaggatcaggttgtgggatgtgagcggtaagatggagaagctgcagaagagccctgcgaggcacatccacaggaagaaagcaaagagggcagcgtgccccacgcagggtggaaactccagagccccaggagccgaggatgaggggcatgcaaagattctgccaaagctcgatattgaacacggagaaaaagtgaactggcttttaagtacaaaaattaaagggaacaaaagcatattagtggctgatcaaacgagttgtgtgtctgtgtatcccctaaatgagcTTTAGGTCCAATAAAATGCATTGCAAAAACCAACAGTAATTTTGGTTTTTTTTGTTTTTTGTTTTGTTTCCTTGCTGACTTATTAAGACACTT

This mutation is a 559 bp deletion beginning at Chromosome 16 position 32,256,492 bp and ending after 32,257,050 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52407 GCT GTG GAG TCT TCA AAA AGC Common A
52408 GCT AGC GCA GGG TTT AGG A Wild type Reverse A
52409 AAG TCA GCA AGG AAA CAA AAC A Mutant Reverse A
52410 Fluorophore-1 ATG AAA CAG AGG AAA CGG AAG G Quencher-1 WT Probe
53390 Fluorophore-2 AGG TCC AAT AAA ATG CAT TGC AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52407 0.40 uM
52408 0.40 uM
52409 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.