Protocol 21512: Probe Assay - Ptprk<em1(IMPC)J> Probe
Version 1.0

Notes

This mutation is a 555 bp deletion beginning at Chromosome 10 positive strand position 28,263,339 bp, TTTTCTATGGCCCTGCCCCA, and ending after TGTTTCCCAACTCCTTCTTG at 28,263,893 bp (GRCm38/mm10).

 

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=  124 bp

Wild Type = 150 bp

Sequence

Wt genomic sequence with the deletion in uper case

ctgaatccgactcctgcttgtctaccatctgaccctaagttagcagttatgtcccatatttctatattgtaaatcagggttaatatcaagttgtagagatgaataaactgtttttgtctagtttatgaaatgttagtaagaaatggatctacgtgatgtcttgactgatatTTTTCTATGGCCCTGCCCCAGAAGACATTTTAAAATTTAATGTTTTCATAGAGTAATATCCTATAATACTCTATGAAATTTTATAGTTGTAATATTTTACAGTTGTTTGATACCCCTGGCTTATCAATGTTCTTAGCTCTGTTATTTGTAATGTCATTTCAGGTTCCTATATGGTTGTGGACTCCTCAAATCATGATCCTGGAGAAAAAGCCAGACTTCAGCTGCCTACCATGAAGGAGAATGACACCCACTGCATTGATTTCAGTTACCTGTTATATAGCCAGAAGGGGTTGAACCCTGGCACTTTGAATATCCTAGTTAGGGTGAATAAAGGACCTCTTGCTAATCCAATTTGGAATGTAACTGGATTCACTGGTCGTGATTGGCTTCGGGCTGAACTAGCTGTGAGCACCTTTTGGCCCAATGAATACCAGGTAATCCCCTGAGATTTTTCTGGTTTGTGCTGTCTTGTATTGGGTATCTTTGTATATATAGATATAAATACTGGTCCTGCAGGAGTAGGATCCCTCCTTCTTGTTTCCCAACTCCTTCTTGaggaggagtttgagtgccttgacacagaaccttctaactatagaaaatatttccatgcatagaaaacagtagtttggcccacaatttgttgt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32631 GAA ATG GAT CTA CGT GAT GTC TTG Common A
32632 Fluorophore-1 CCT GCC CCA GAA GAC ATT T Quencher-1 WT Probe
32633 Fluorophore-2 AGG AGT TTG AGT GCC TTG ACA Quencher-2 MUT Probe
32634 CAG GGG TAT CAA ACA ACT GTA AAA T Wild type Reverse A
32635 ACA ACA AAT TGT GGG CCA AAC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32631 0.40 uM
32634 0.40 uM
32635 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.