Protocol 44099: Probe Assay - Lrrc63<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=  93 bp

Wild Type = 100 bp

  
>chr14:75123208-75123307 100bp CCAATCCTGTCTGGGTGAGT GGCATGGATGCTCTCACAA

Sequence

Wt Sequence (deletions in lower case):

CCAATCCTGTCTGGGTGAGTGCAGGATCTGGGgagttgattccagactacctggatcgccaatccctttggtaattcattcttgtgagagcatccatgccacttagccatggaagtcataataccctatggtttgacattttgaatatctatctatctatctatctatctatctatctatctatctatctatctatctatctatttattagctgttcagagaaccaccagaacaataggcccagatgcccatgtcttacgaggggaaggctttaaggctgttgcagcaacacgctctgaaacagtactggcgctgaccaccctggccataataaattgccaaatatatggaagaaatgcacttaatcttaaggtatgttagacctccttaatagtcatgcccattggtattatcagttatttttaccttttcagtttctcaatctaggttagttttcattacattgaaggagcttttcctattaccgcagacacttcccaggaatacatgcaaaacatacctccaaacttaacttatctaagtttatcttcagttcaccaataaagtttaaaaggttgctgcgatccagagaagtcatactgtttttatttttttttccctcatttattctgggAAGCAGGATTGAAATTGTTCATGTTTCTAAATTACCAGAAGCTTTGGACCTTGTGTCAAGT

 

This mutation is a 602 bp deletion beginning at Chromosome 14 position 75,122,674 bp and ending after 75,123,275 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64771 CCA ATC CTG TCT GGG TGA GT Common A
64772 GGC ATG GAT GCT CTC ACA A Wild type Reverse A
64773 ACT TGA CAC AAG GTC CAA AGC Mutant Reverse A
64774 Fluorophore-1 TTC CAG ACT ACC TGG ATC GC Quencher-1 WT Probe
64775 Fluorophore-2 CAG GAT CTG GGA AGC AGG ATT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64771 0.40 uM
64772 0.40 uM
64773 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.