Protocol 45381: Probe Assay - Diras2<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

>chr13:52508194-52508288 95bp GCTGGAGTTCTGAAGCAACA TCAAAACGAGGGAGCTCTTG

Mutant=  99 bp

Wild Type = 95 bp

Sequence

Wt Sequence: GCTGGAGTTCTGAAGCAACATGCCGGAACAGAGCAACGAcTACCGGGTGGCGGTGTTTGGGGCAGGCGGTGTTGGCAAGAGCTCCCTCGTTTTGA

Mut Sequence:

GCTGGAGTTCTGAAGCAACATGCCGGAACAGAGCAACGActGAGAGGCCTTCGTGCAGGAGCTGCTGCTCTCGGGCCTCTCTCTTCCTCCCCTCTGTGA

 

A 576 bp deletion beginning at Chromosome 13 position 52,507,673 bp and ending after 52,508,248 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65466 GCT GGA GTT CTG AAG CAA CA Common A
65467 TCA AAA CGA GGG AGC TCT TG Wild type Reverse A
65468 TCA CAG AGG GGA GGA AGA GA Mutant Reverse A
65469 Fluorophore-1 AGC AAC GAC TGA GAG GCC TT Quencher-1 MUT Probe
65470 Fluorophore-2 CGG GTG GCG GTG TTT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65466 0.40 uM
65467 0.40 uM
65468 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.