Protocol 33154: Probe Assay - Ints3<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

>chr3:90413194-90413273 80bp GGGGACTGAGAGGGAAATGA GGAGGGAGGAGCGTATGGTA

Mut= 82 bp

Wt= 80 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 AGAACAATATCATCCTCTCTGGTTTGGGGGGGAGGGCAGCAGTTCTAGAACATTCTTTCTCTCTCAAGGCAAAGGGAAAATGTTTACATCCTAAAagAGAGAACGGTTTATTCTAGCATTCAGGGCAATAGTGGTGGGAGAGTGGACcatcgggatgactgaagagatttattcgattagctgagttgctaattagagctactgtattctgattcttccccctcccaccccttgaaatcaggtggcgatgttacagccaaaaatatctggttggcagaaagtgtcctggatatcctgacggagcagaggtagagtctcccatgaagggtggggctgtgaggggtgtgagtatgggacatgggaaagaaaggctggtggctcagagcactctgaggccaggggaaggtgtggcaggcctgatggtggcagatggagaactaaggtcaaaagatggaggaatgggaaagaaggcgcttgctaaaatatctcagagcccagaaaatctggtctcatctgataagggtttatttttgacccagacctccttggaatgtttatgtttggggggactgagagggaaatgagctcagcagaggggccagccaacCACACGAGCCTGGCAGGTACCATACGCTCCTCCCTCCCAGCCTCAGGCCAGCACATGCTCTTTTTTGGAATGTCACAGGGCCAATGTAGGTGACTAGGGAGCAGAGAATCCTGCTATAAAGTGACATGGGGCCACATTTGAACTTC

This mutation is a 460 bp deletion beginning at Chromosome 3 position 90,413,231 bp and ending after 90,413,690 bp (GRCm38/mm10). In addition, there is a 2 bp deletion (AG) 50 bp before the exon deletion that will not alter the results of the exon deletion. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40762 GGG GAC TGA GAG GGA AAT GA Wild type Forward A
40763 GGA GGG AGG AGC GTA TGG TA Common A
40764 ACG GTT TAT TCT AGC ATT CAG G Mutant Forward A
40765 Fluorophore-1 CAG AGG GGC CAG CCA Quencher-1 WT Probe
40766 Fluorophore-2 AGT GGA CCA CAC GAG CCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40762 0.40 uM
40763 0.40 uM
40764 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.