Hosuk:LabNotes/2014-1-19

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NCIH1975 : Mutation Detection

Padlock Probes

  • Padlock probes selection
  • 1)EGFR T790M
    • Wild : detected by dcProbe1
    • Mutant : detected by dcProbe3 or dcProbe4
  • 2)EGFR L858R
    • Wild : detected by dcProbe4
    • Mutant : detected by dcProbe2


Procedure

  • Experiment Plan
    1. Anneal Padlock probes – 2 probes (Preheat probes at 70C for 5min, incubate at 50C for 1hr.)
    2. Add SNL mix (Ampligase + Stoffel enzyme mix)
    3. Incubate 50C for 4hr.
    4. Anneal Primers for 2nd RCA
    5. Run RCA reaction for overnight(12hr.)
    6. Anneal dcProbes, and Image