Protocol 35738: Probe Assay - Piezo2<em2(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

>chr18:63030174-63030281 108bp AAAAGAGGTATCTGTTGCATGG CTTTTGATAGCACCGAACCA

Mut= 96 bp

Wt= 108 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAGATGTGTTCTAGAAGGTTCTAACAAATTGAGAGGTCATTGTCTCATCCTTACTCTATCTGCCTCACTCTACCTGCCTTGGCTTAGTTACTCATACAAACTCTTTTCACCCGCAAACGTCTCCACTCTCCTAGGACACTCACAATTCTGTACAAGCTTCCTTAGACATAAATCCCTTGgctttccaacgtcaagggattgaatgttggaagggacactgtaagaaagacagccagttggcccacccaagatgctagctagcctcagctgtcatcttctcttcttctaacagtgtcatggtctatgggacgaggatgacattgttgacagtaacacagacaaggaaggatcggatgacgagctgtccctcgatcagggcagaagaggctcctcagactcactgaagtctatcaacctggctgcctctgtagagtctgtccacgtgaccttccccgagcagccagcggccatcaggagaaaacgctcctgcagtagctctcagatctccccacggtccagcttctcttcaaataggtccaaaagaggtatctgttgcatggccattaggttgaaatgagccccacatagtctcattgATCTGGTGCCTCTGCATCTGGGGAGGGAAGTGGTTCGGTGCTATCAAAAGACGATGTTGTGACATGGAAGAAACATGCTCTGGCATATGACAGACCCAAGTTCAGAACCTGTTTCATCTGTATGTAGCTTCATTGTTCAATATGTGTGAGTCCATTCAGGGACCAGGATTTGGCCAGCACATAACCTCATAACCTA

This mutation is a 417 bp deletion beginning at Chromosome 18 position 63,030,224 bp and ending after 63,030,640 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46902 AAA AGA GGT ATC TGT TGC ATG G Wild type Forward A
46903 CTT TTG ATA GCA CCG AAC CA Common A
46904 GGA CAC TCA CAA TTC TGT ACA AGC Mutant Forward A
46905 Fluorophore-1 AAA TGA GCC CCA CAT AGT CTC A Quencher-1 WT Probe
46906 Fluorophore-2 AAT CCC TTG ATC TGG TGC CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46902 0.40 uM
46903 0.40 uM
46904 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.