Protocol 37920: Probe Assay - Zdhhc22<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

>chr12:86988091-86988183 93bp TCCTGCCCACTTCAATCAG AAACTTGGGGTTGGGCTAGG

Mut= 97 bp

Wt= 93 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower casen):

CCTCCCTGTACCTCTCCAGACTCCAAGACAACAAGGACTTAGCCAAATGCAGTAGGTCTAGCCAGATGCTGACCAACTGTGGTCTCTGGGTCTCTAACCTGCTTGGTTCCAAACTAGGGAAACTGGAAAGGGTGATCCAAGGGGCAGGGTTTGGGAGATTTGTGTCCAGCCCTTCgaaccttggacaatgctgtaaggcttcctctctccttgtgccccttaggaatgtgatccaggatgctggccttgcggctgctcaacgtggtagcccccgcctactttctttgcatttccctggtgaccttcgtactgcagctcttcctcttcctgcccagcatgcgtgaggaccccacagccaccccgctcttctcgcctgctgtgcttcacggggcgctcttcctgttcctctcagccaatgccctgggcaattacgtcctggtcatccagaactccccagacgacctgggcacctgccaggggaccatgtcccagagacctcagtgcccaccgcccagcacccacttctgccgagtgtgttcccgagtcacgctgaggcacgaccatcactgtttcttcaccggcaactgcatcggcagcagaaacatgcgcaacttcatcctgttctgcctctacacctctctggcctgcctttactccatggtggctggagtggcctacatctcagctgtcctttccatctccttcgcccaccccctggccttccttacgctcctgcccacttcaatcagccagttcttctccggtgagtgggccatggcaggCAGGTTCAGCAAACAAGACCTCCTAGCCCAACCCCAAGTTTTTCAAATAAAAACCACAGGATGCTGGACTCAAACAGTCAGATAGACAGTTGTTTGGTATGACTATGTTCCAGTATTCCAGGAGGCATACTAAGAGAACAGATTATAATCTATCAGCAATTAGGATGAACTGGGCA

Thsi mutation is a 607 bp deletion beginning at Chromosome 12 position 86,988,132 bp and ending after 86,988,738 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52511 TCC TGC CCA CTT CAA TCA G Wild type Forward A
52512 AAA CTT GGG GTT GGG CTA GG Common A
52513 AAA CTG GAA AGG GTG ATC CA Mutant Forward A
52514 Fluorophore-1 CCA GTT CTT CTC CGG TGA GTG Quencher-1 WT Probe
52515 Fluorophore-2 CCA GCC CTT CCA GGT TCA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52511 0.40 uM
52512 0.40 uM
52513 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.