Protocol 41294: Probe Assay - Opcml<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 = 98 bp

Wt = 103 bp

Fam = Mut

Hex = Wt

>chr9:28813184+28813286 103bp GCCTGAGAGGCAAGTGTAGT CAAATATCCATTCGCTGTGA

Sequence

Wt Sequence (deletions in lower case):

CAGCAAGTGGCAACTGTGACGAATTTAGAATGTATGTGACTGCCTGAGAGGCAAGTGTAGTATCTTCACATTTAAATGTAAcatcttcctaaaatgaatggttggttgactaaggggaggggtgtcacagcgaatggatatttgtcttctctggaattaagttttaatttatttattttttctgtctgtcttgcttttatttctccagaccctccctatatatctaaagccaagaacactggtgtttcagtgggtcagaagggcatcttgagctgcgaagcctctgctgtcccgatggctgaattccagtggttcaaagaagataccaggtaccttgaaggaactgggggagctctgacacagagctgattaatcatgcatcccccagaaggagggtcacaaactccaggggtaaaataaacaagataaaaacatgctataacctccttttaatggagtcttcttttagaatcagaagtgagaaagaaaaaaataaatagagggagctgagaactcaacaaaATGGATTGTCCTTGTTTAAGAATCTTGGATTACAAAGTTTGGAGCTGAGACAAAAGGA

This mutation is a 441 bp deletion beginning at Chromosome 9 position 28,813,224 bp and ending after 28,813,664 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
57951 GCC TGA GAG GCA AGT GTA GT Common A
57952 CAA ATA TCC ATT CGC TGT GA Wild type Reverse A
57953 TCC TTT TGT CTC AGC TCC AA Mutant Reverse A
57954 Fluorophore-1 AAT GAA TGG TTG GTT GAC TAA GGG Quencher-1 WT Probe
57955 Fluorophore-2 AAT GGA TTG TCC TTG TTT AAG AAT CTT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
57951 0.40 uM
57952 0.40 uM
57953 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.