Protocol 34663: Probe Assay - Ppp1r12b<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

Mutant=  74 bp

Wild Type = 86 bp

Sequence

wt sequence with deletion in lower case

ATGACAACAACAAACCTGTGTTTTCCCTACTTGCTGTGATAAAGGCAGAGAACAGTATCTGCCTGCACCTACAAATACACTTTCCTGTATCATCCAAAGCCATTTAGAAAACCACATGAGAACATTTCTAACACGCTTTCCTTTATAACACAATGCTGCAATAGCTTACCCATTtacagcactgtagagatcatttcatagttaatcccattaatactgaaaataatatctaatagtgcttctaagttggaagtcatatttcagacacttactaggaaaaatgagtagtgcatcagaactgctagtcactgctgttgtccagagcagtgtctggacaaaatagaactcagtaaatgcttgttgaatgaatcaatgggcttttcttttcaggcctgtattgatgaaaatttggacatggtgaagtttctggtggagaacagagccaatgtaaaccagcaggacaacgagggctggacacccctgcatgcagcagcttcctgtggttatctcaacatagcagagtgagtgagcggcgttgtgggtttgatgcacattcctcctctgcttcccagaaatggaatttcacaaatagttataaatttctctaaattacaaaagcagaaaattgactattaagagctagctgacactgtgctttgtatctcaatgcacagttcttagctccaatttttgtttgcttctgtttttgtatagttcataatttctggactattcagaggcagcatcagcctggcATGCTGTCTGGCTACTTTCCAAACAGCAGAACACAAGCATGGGAGTTGTAGTTTGTTTGTTTGTTTTTTTAACTTTTCTGTAAACCTGCTTTTTGTTTGTTTGTCTTTATAAATATTATTGCAGTAAATAGCAAATTCTTAGCTGGAACAGATGGAACCAAGGCAAAG

 

This mutation is a 581 bp deletion beginning at Chromosome 1 position 134,902,144 bp and ending after 134,902,724 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44171 GGT GGA GAA CAG AGC CAA TG Wild type Forward A
44172 TGT TGA GAT AAC CAC AGG AAG C Wild type Reverse A
44173 CAA CTC CCA TGC TTG TGT TC Mutant Reverse A
44174 CAC AAT GCT GCA ATA GCT TAC C Mutant Forward A
44175 Fluorophore-1 ACA ACG AGG GCT GGA CAC C Quencher-1 WT Probe
44176 Fluorophore-2 CTG TCT GGC TAC TTT CCA AAC AGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44171 0.40 uM
44172 0.40 uM
44173 0.40 uM
44174 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.