Protocol 43928: Probe Assay - Tjap1<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

>chr17:46260812-46260929 118bp CTGCTGCACTCTCCTCTCAG GTCCCCACAATCCCTGACAT

Mut= 104 bp

Wt= 118 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TCAGCTACCAGACGCACTGAGGCCTTGGAGCGGGAGCTGGAAATAGGACAAGACTGCCTAGAACTGGAGTTAGGACAGAGCCGTGAGGAGCTGGACAAGTTTAAGGACAAATTCCGCAGGTGCGGAACAAGGGGCCAATCGTCTCCCATCCGTGGGAGCACCTTCCTGTCCATACGTTAGCTGAAGAGCCTCAGGTCCccctgactttatttagtaacccagatcttggaaggagagggaagttcctagactcccatgtggaggagggttggtggggcctgaggaacaggccttaaccagggctctgcctttctctgccttgttggcatgtgtaggctacagaacagctacacggcatcccagcggaccaaccaggaactggaggacaagctgcacgcactggtactgtgctgggagggtggtgtggccacaacatagcgcgggctaggacttccgggggggatgccactcaccctggggcctggggaggagagggaaggcagggcagtggtggggagggcgcggtgtgctgcgccaggcgggacagccctaatgctcacactgtctctcttctctctccgtctccctccctcatgctgccaccgctgctgctgcactctcctctcaggcctctcttagccacagctggatttttgcagttgcgtcTGAGTTTGTGTGTTTTCCCTGGTCCTGGCCTCCTTGGTCCATGTCAGGGATTGTGGGGACCCTGGCCATTTCCCTCTGCCCCTTGACCACACCCTCTCTTTTCCTGTCATGTGAGACCCAGAGTGCCTCCCTCCTGGGAAAGGGCTAGGGCTGAGGTTCTGCTCATGGCTCTGGCAGAGGCACTGAGTGATAGCCCTCAGCAGTCCCAGCCTTGGCTG

 

This mutation is a 468 bp deletion beginning at Chromosome 17 position 46,260,872 bp and ending after 46,261,339 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64364 CTG CTG CAC TCT CCT CTC AG Wild type Forward A
64365 GTC CCC ACA ATC CCT GAC AT Common A
64366 GGA GCA CCT TCC TGT CCA TA Mutant Forward A
64367 Fluorophore-1 CTC TCT TAG CCA CAG CTG GAT T Quencher-1 WT Probe
64368 Fluorophore-2 AGC CTC AGG TCC TGA GTT TGT GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64364 0.40 uM
64365 0.40 uM
64366 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.