Protocol 37647: Probe Assay - Thoc3<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

>chr13:54468178-54468260 83bp GGGACTAATGGGGTGGTAGA ACCTCAACCAACCTGTCCTG

Mut = 81 bp

Wt = 83 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

AGCCCTGGTGACGAGTGGCTAGGGACTAATGGGGTGGTAGAGCCcctagggtggagaggaaggctttatttgaaaatgatagcgcaggacaggttggttgaggtggtggtggaaaaagaaagaagggaggccaggaagggccaatgtcaaagctcagagaggttcatgggggggggggggagggggtgttgtagttttggaaaaagaattagatttctgttttgaattatccagatctttgctggtttcaaggcaccactgtttggataaaagtggcatctggattctgttcttttaaggtcaaagaaaacaactaccggggccacggagatagtgtggaccagctttgttggcacccaagcaaccctgacctctttgtcaccgcatctggagataaaaccattcgcatctgggatgtgaggactacaaaatgcatcgccactgtgaatacgaaaggtgagttcagagagagggggttctggaggaaaagctagtgtgatctgcagcaagcatctttgtagcaagtgcaggttatcaggatgggttgtatgtgtctgtctggagaagacaggaacagaacagagcacttttctggcaggctgtagatgtataggagagcagtggaaactatacaagagtagtatgtgcagaggcgtggagagtttgggagattcaggcaggttacaatggctgagaaaccatggGCGATCTGGAAAGTTGTGCGTTTTGTTTAAAGGCTGTGTTCAAAGAAGCAGTGTGGTAGTTTGAGTAGGCTTGGCCCAGGGCATGGCA

This mutation is a 660 bp deletion beginning at Chromosome 13 position 54,467,578 bp and ending after 54,468,237 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51844 GGG ACT AAT GGG GTG GTA GA Common A
51845 ACC TCA ACC AAC CTG TCC TG Wild type Reverse A
51846 TAC CAC ACT GCT TCT TTG AAC A Mutant Reverse A
51847 Fluorophore-1 AGG GTG GAG AGG AAG GCT TTA Quencher-1 WT Probe
51848 Fluorophore-2 CCG CGA TCT GGA AAG TTG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51844 0.40 uM
51845 0.40 uM
51846 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.