Protocol 35446: Probe Assay - Dipk1c<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

>chr18:84730298+84730444 147bp AGAACACGCTAGGCTTGGAG GATCTGGAAGCAGGCTGAAG

Mut= 150 bp

Wt= 147 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CTGTTCTGTGCAGATGCAATATTTAATCAATGTTTCCACTAGGCGTTGGCTGGGGAACTCACAGAACAAGGCAATCACCTGAACCCGCCTCCCGGGTGGCCTCCAACACTCTCATGGGCATTTTCTCTCCTCAGCTAATTACAAAGTCCTTTGCCCCCTATTCCTGCCCTgcagtgaccctctgttccttttgttccagtgccaggactatcgccgtggctggctcacgggcgccctgtgcgaggacctgtgtgtgggcggggagctgctgtaccaacgctgcctgtattacgagcgtggtaagaaggtgctgcaggctcagtggcgtggccggaccgttgtcctcaaatccaagagggaggccttctccagcttcccgcccctcactctgctggaggaggaagcaggggcaggtgcccccggcatccctgaggctgagctgctcctgatggtggccggggaggtcaagaacacgctaggcttggagctgcccaacaacagcatagccccgctatggcctgccaggcagggccctggctggcggcaacagctggccagtgcgtggtcactgctccaacaggaggaatacgtatacttcagcctgcttccagatctgagccgccacatcctaccggtgctgggatcctgcggtcacttttatgcagtggagtacttggctgctggtagccctcaccacaaggctctcttcccactggatgatgctggccaagcacaggctattagccacattgccctcagcttcctggacatggtgagccatttcgacagtgacttttcccaccggctccacctctgtgatgtcaaacctgagaacttcgccatcaagagggactttacggtgagtggaagcaggctgccatgtttgtggtaagagaagagattctggtctgtgtaggaggaagtgaattcttggcctctggaaggaagcccatgcaactttgggcagccttgtggttcctggtgattcccgttccagaccacttccttgccaggttgacaaaagggtttaagaaaatgcatctccaacctcaagaggagaggtccagtccagtgaagaagcccacaggaggcagagacaggcagggggcggaggagacatctcacttccttgaggGCAGCTATGAGGTCTAGTTAGTGCTGCTACAGGATCTGTGGTCTTCTGAGGACAGTCTTCCTGGGCTGCAGATGGTGTGCTGTTCTTCCAGGCCAGGCATTCTGTGTCCACAGAACCAGTGTGCAGAGAACAGTCATTGCCAGCCGACATTACTCTTCTGGAGAGATTCT

This mutation is a 965 bp deletion beginning at Chromosome 18 position 84,730,001 bp and ending after 84,730,965 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46077 AGA ACA CGC TAG GCT TGG AG Wild type Forward A
46078 GAT CTG GAA GCA GGC TGA AG Wild type Reverse A
46079 ACA AGG CAA TCA CCT GAA CC Mutant Forward A
46080 AAG ACC ACA GAT CCT GTA GCA Mutant Reverse A
46081 Fluorophore-1 CTG GCT GGC GGC AAC Quencher-1 WT Probe
46082 Fluorophore-2 AGT CCT TTG CCC CCT ATT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46077 0.40 uM
46078 0.40 uM
46079 0.40 uM
46080 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.