Protocol 36585: Probe Assay - Dpp8<em1(IMPC)J>-Alternate 4
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=  112 bp

Wild Type = 109 bp

>chr9:65042263+65042371 109bp GCCATGTCTGGTGAGAACAG AAAGATCCAAAAGGGGCTTC

Sequence

Wt Sequence: AACTTTGGCAGTCTTGTTTTTCTTACTCAGGTTTTTTGTTTTGTTTCATTGTTTTTTGTTTTTTGTTTTTTGTTTTTTTTTTTTTGTTTTGTTTTGTTTTGAGACAAGGTTTCATTTAGCCTAGGCTGGCTTCAAAATCCTGATTCTCCTGCCTCTACCTCCTGAGTTGTGGGGTTATAGGTGTTTGCTACCATGCCTAGCTTAAAACATTGTTTTTATTCAAGGAAATTTCcacggcatgaagcatgtgtataccctacttaataacatcattttggttcctgtggaatggaaaatattataatatgtagaagacattatgtatttacgactgttgtaataaggacaatattacaaaataataatgtattttttgtttttgtttttttccttatagccatgtctggtgagaacagagaaaatacactattttattctgaaatccctaaaaccatcaacagagcagcagtcttaatgctttcctggaagccccttttggatctttttcaggtaggaacaagtattcttagtcctatgatttggtgttatttcttcatttatttcagggcaaatgtttttCTAGGGTTTTATATTTTAAGTAGATGTGAATAAAAAGGAATTTAAAATGTGTAATTGTTTTTACATGAATGACTTTAAATGACTAATTTCCTGTATCTAAAATCATAGGATATTGTAATCCAATAATTTTGGTGTTTCATTTGTTTGAAATGTTTTGGTTTGTGTTTGTGAATGTTTTGCTGGCATGTCTGTCTGTGCACCATACACATGCCTGGTGTGCATAGGCATTAGAA

 

Mutant Sequence:TCAAAATCCTGATTCTCCTGCCTCTACCTCCTGAGTTGTGGGGTTATAGGTGTTTGCTACCATGCCTAGCTTAAAACATTGTTTTTATTCAAGGAAATTTccTAGGGTTTTATATTTTAAGTAGATGTGAATAAAAAGGAATTTAAAATGTGTAATTGTTTTTACATGAATGACTTTAAATGACTAATTTCCTGTATCTAAAATCATAGGATATTGTAATCC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46332 GCC ATG TCT GGT GAG AAC AG Wild type Forward A
46333 AAA GAT CCA AAA GGG GCT TC Wild type Reverse A
48449 Fluorophore-1 ATT CTG AAA TCC CTA AAA CCA TCA Quencher-1 WT Probe
49159 Fluorophore-2 ACC ATG CCT AGC TTA AAA CAT TGT Quencher-2 MUT Probe
49160 TGT GGG GTT ATA GGT GTT TGC Mutant Forward A
49161 TCT ACT TAA AAT ATA AAA CCC TAG GAA Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46332 0.40 uM
46333 0.40 uM
49160 0.40 uM
49161 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.