Protocol 41355: Probe Assay - Arl14<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

Mut = 117 bp

Wt = 122 bp

Fam = Mut

Hex = Wt

>chr3:69223001+69223122 122bp CACTGGAGAAGGGCTGGAT CCATTTCCACTTCATCATTGTC

Sequence

WT Sequence (deletions in lower case):

GAAACGATCTTTAAGGAAGAAAAAGCCTAAAATGGGTCTGCTGAATTCTAAAAACCCCCAAAGCAAGCAAGCCcacattcttctcctcggacttgactcagctgggaaatctactctgctttaccggttaaagtttgctgagactctctcaaccatcccaaccatcggcttcaacgtggaaatggtccagctgcagagcagtctcacactcaccgtgtgggatgttggaggacaggagaagatgcgaacagtctgggactgctactgtgagaacgctcaagggctgatgtacgtggtggactgttccgaaggcaaaaagcgactggaagactctcgcaaagagttcaaacacattttgaagaacgagcacatcaaaaacacaccggtcgtcatactggccaacaaacaggacttgccgggagctctgagcgccgaagacatcaccaggatgttcaaggtgaagaagctgtgcagcaaccggaactggtatgtgcaaccctgctgtgcggtcactggagaagggctggatgacgggttcaggaaattaaccgagtttctgaaaagctaccgaaggacaagagagaccttagcaatcttCAAGCAGAAATGAGACAATGATGAAGTGGAAATGGGAAA

This mutation is a 524 bp deletion beginning at Chromosome 3 position 69,129,897 bp and ending after 69,130,420 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
58118 CCA TTT CCA CTT CAT CAT TGT C Common A
58119 CAC TGG AGA AGG GCT GGA T Wild type Forward A
58120 GGG CCA AAA GAA ACG ATC T Mutant Forward A
58121 Fluorophore-1 ACG GGT TCA GGA AAT TAA CCG Quencher-1 WT Probe
58122 Fluorophore-2 CCA AAG CAA GCA AGC CCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
58118 0.40 uM
58119 0.40 uM
58120 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.