Protocol 37253: Probe Assay - Cpd<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

>chr11:76840606-76840718 113bp TGAAAGATCCTGTGGAGGTT CCACCATGAAGATTTCCAGA

Mut = 114 bp

Wt = 113 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GAACTGGAGGATAGAGTCAGTGCCTAGCATGTGCCCAGGCCCTGAGTTCATCCTCAGGACTGATTAAGTGAATGATTGATTGATTGAATGAATGAATGAATGAATGAAAAAAGAATAAAGGAAGAAAAAACTGAGTTTATTTATGAAAGATCCTGTGGAGGTTTTAAATGCTTGGTATTGTCTGTGCCTTctaagggtgatatgtaatttgattatgttttcaaaggtttgtgctttctggaaatcttcatggtggctcagtggtagcaagctatccttttgatgattctcccgagcataagaccactggactctatagcaaaacctccgatgatgaagtctttagatacttggcaaaagcttatgcctctaaccaccctataatgaaaactggcgaaccccattgtccaggagatgaagacgagactttcaaagatgggatcacgaatggcgcgcactggtatgacgtggaaggtatgcaaagcactgactgttgcatttccttttgttggtttagctttcttacatgtattctgaatgcaggaggaaatgtgcaatttcacttcttaaacaggttatatctttgtatgtctgaagtttccaaaactacagcaaaatttaaagacgaccacagaaatatgtactatctccagttgacctcccctgctgatctCTTAACCCATATGCTCCACCTCCCCGCCTACACATGCCCCCCCCAATTCTTCCCCAGACCACTCAAAAATATGTAGCTGACAGATCCCTTAAGTCCCTCAGCACTTGTTCTTCCAAGCACCATTCCCCCACCTGAGAG

This mutation is a 483 bp deletion beginning at Chromosome 11 position 76,840,189 bp and ending after 76,840,671 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50890 TGA AAG ATC CTG TGG AGG TT Common A
50891 CCA CCA TGA AGA TTT CCA GA Wild type Reverse A
50892 TTT GAG TGG TCT GGG GAA GA Mutant Reverse A
50893 Fluorophore-1 AGG GTG ATA TGT AAT TTG ATT ATG TTT TCA Quencher-1 WT Probe
50894 Fluorophore-2 CTT CTT AAC CCA TAT GCT CCA CCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50890 0.40 uM
50891 0.40 uM
50892 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.