Protocol 37759: Probe Assay - Ccdc166<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

>chr15:75981236-75981348 113bp TTGAAGAACCTGGAGCAAATG TGACTGGTTGACCCGATG

Mut = 120 bp

Wt = 113 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GGGCACTTGGAAGTTAGTTCAGTGTCTTGAAGCTGAAGTTCTGCCTGACCTGCGACACTGTAACTGAAGAATCATGCAAGGATATAAGAAACCTTAGGGAATGAATGATTGAGTCAAGCCGTTGccagagcaacagaatgggatgaggtcgaggcactgcaaccaagttctgtctccatagaggttgttcaagctggacatcaacattcctagagctacgcaccaggatgcgaaagctgtggtctggagggaacgcttatatacagattggccacggtgctctctgatccttaatcctgcctgctctgaacctcataatcagaagacacacccttgaacctagggtgcagttctccagaattttcgggttaagaagggagagtaactgagctgggggagcgggtggagcacagcgctctcatgagtccagttggcttgggcctcaagccatggcgcccaagaagaagcgcggacaaaacgcggggcgcaagcaggaaactacagagcaaccgctctcggagcgcgcgcagtacctacagcgcgagtacgcgttgctctcagaaagcctggtggcctgtgagcagcggatcgacgaggtgctgcaggacaacgagttcctgaacagagaggcgcagcgcctgcgcgaagagaaccggctttatgtcggttacgtgagcgcgcacgctctgcgctgcgccaacgccgtcgtgcgcgtcgaagaccagaaccgcatggatctggcgcagatccggtggcagcgagcggagctagcgtccttctaccacggccgtgaggacggggtgcgcgctcagctgcaggaaatgaagaagcgcgcggaaaacatgacgcagcgggtgcaggagctccagccctacaaggccagtgcgcgcgccccccgaggaccgggcgggctccagagggatgggcgcgctccctaccggtgcgctgatcggcgccccctgctccccaaccctcaccccaccccatgtaggagctgcagctggagcagctggcgcgaatccggaccctggagcgcgagctgctgcacatgcgcgtggagcacacgcagctcctccacggggtgaagcgacgcttcctagatgagaagacggctttcgagcgggaggcgcggctgcaagtgcagtccctgacgcgccgctccgagcgggaggccgcacgttccctcatttcccacgcacaggccctcaaggcggacaacgggtatctccgacaagaactcatgagacttctccagaggacccagttactgcaggacatgcgacagcagctgctagagcagcgggagcagctgcgccgggagcatgtggacttgaagaacctggagcaaatgcacggctggttgcaaaggggccccggggggccaccgctctggcagccaccccaaagcctccaacccagcttgcgcatcgggtcaaccagtcacgttttaaaagcccaggctacgccccagtcagttccaacctcccggggccaatccatggatctgtcacaccgaccctccaagactccatctatattgtccagtgagtctcttccgggcctatctcagaaggctgggtcagtgattgctctccggagtccatcacatccaggatctcgagtctcatctctgactccatcacgcaaggactcgcgtgtctcatccgcgaccccgtcacgcaagggctccagggttccatccatgacctctggtaccttgcggtcgcgcgagggctctcggatctctccacagccctccttgcgtgaggtctctccagaaattgacacacctgcaaagtccagttccaagcttcccacagccctgttggaagatcaggcccctctcagcccacagttggaggaggcagaaaacgctgaagacgacacagaaactgtcttggagcaagcctgaacccacacgaggaggcaggccagtagggcatagggtggaggacaaaccttggaagatttcttggtgcatagatatgttaataaaagtgttaaataagctctgtgacgctctgacacttgcctgggtcgatgcccttgtatttcaagtgagcagaggcctgctattcgagctacagcagagctggttgtatccttggctgctggaattcgatgttcttccaccaacagccagcaaggcaggcaggcttagggaaagttgggtagtgtaatttcagcagcagagggcagattctgttctcagcacagggagaagttactgggcttttccaatgctttatataatacgcacacacacatatgtatgtaggtatgtatatgtatatatgtatatacattccaatttagggaacactgcaagaattattaggaattcccattgccacccatctagatgaacagggatgcatgttatatatttggtactttcagccatccgggattctgttggagacagttatgccccttaattttaaatatatccctgtgtttactgaataggactttgacctgttttcttccggtgctgggtgttgaacccagggctacctcctgagctacagcccaagccctgtcccaactttcttcaagacatgattaaaattaggaaatgcaccttggagaaaatactgtaaactaaatcatactggccaaattgtccctgtggtgtctgtagctagctatggatcttacctgggtcacatcttacattccattgtgctggtctgtcctttcctttatagaaagggtctgtataggtcaggctggccttgaatccaagagatcctcctgcctctgtctcttcagtgctgagattaaaggcatccaccaccacctcagccccgttgtcttgtctttttttttttaaacttctttttaaagatttatgtatatgagtgcactgtaactgtcttcagacacaccagaagagggtgttgggtcccattacagatggttgcgagccaccacgtggtcaccagggattgaactcaggaccactggaagagcagtcagtgctcttaaccgctgatccatctctccagccctctttgtcttaatgtacaagaccagtcGTTAGAGCACTCCTTAACTGAGTTTTGGAGTCTGTTGGCTTTGAGCACCTTTCCTGTCCAGGAAGTCCAGGGCAGTTGACACCTCCTGACCCAGG

This mutation is a 2917 bp deletion beginning at Chromosome 15 position 75,979,638 bp and ending after 75,982,554 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52088 TTG AAG AAC CTG GAG CAA ATG Wild type Forward A
52089 TGA CTG GTT GAC CCG ATG Wild type Reverse A
52090 TGA CCT GCG ACA CTG TAA CTG Mutant Forward A
52091 AAG CCA ACA GAC TCC AAA ACT C Mutant Reverse A
52092 Fluorophore-1 CCA AAG CCT CCA ACC CA Quencher-1 WT Probe
52093 Fluorophore-2 AGT CAA GCC GTT GGT TAG AGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52088 0.40 uM
52089 0.40 uM
52090 0.40 uM
52091 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.