Protocol 34374: Probe Assay - Cmya5<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

>chr13:93098350-93098439 90bp TTCACCATAGGTTATCAGATGAGG GAACTGCCACCATGGAAAAG

Mut= 93 bp

Wt= 90 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence :

 ttcaccatagGTTATCAGATGAGGATGAGGAGGGCAAGACCAAGCAGGAGTGTATCGTCTCTGACCCTTCCTTTTCCATGGTGGCAGTTC

This mutation is a 9281 bp deletion beginning at Chromosome 13 position 93,089,133 bp and ending after 93,098,413 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43445 TTC ACC ATA GGT TAT CAG ATG AGG Common A
43446 GAA CTG CCA CCA TGG AAA AG Wild type Reverse A
43447 TGT GAA AGC AAA CCC AAT ATC Mutant Reverse A
43448 Fluorophore-1 AAG ACC AAG CAG GAG TGT ATC G Quencher-1 WT Probe
43449 Fluorophore-2 AGG TAA ACC AAC TTA ACA CAC ACA AAT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43445 0.40 uM
43446 0.40 uM
43447 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.