Hosuk:LabNotes/2014-1-19: Difference between revisions

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====Procedure====
====Procedure====
*Experiment Plan
*Experiment Plan
*#Anneal Padlock probes – 2 probes (Preheat probes at 70C for 5min, incubate at 50C for 1hr.)
*#Anneal Padlock probes – 4 probes
*#Add SNL mix (Ampligase + Stoffel enzyme mix)
*#*EGFR T790M(Wild, Mutant), EGFR L858R(Wild, Mutant)
*#Incubate 50C for 4hr.
*#*50nM each in 2x SSC + 30% Formamide (2uL each)
*#*Preheat probes at 80C for 5min, incubate at 50C for 4hr.
*#Add SNL mix (Ampligase + KlenTaq enzyme mix)
*#Incubate 50C for 20hr.
*#Anneal Primers for 2nd RCA
*#Anneal Primers for 2nd RCA
*#Run RCA reaction for overnight(12hr.)
*#Run RCA reaction for overnight(20hr.)
*#Anneal dcProbes, and Image
*#Anneal dcProbes, and Image


[[File:ReactionMix_Ampligase_KlenTaq.png|550px]]
 
[[File:ReactionMix_Ampligase_KlenTaq.png|600px]]

Latest revision as of 00:38, 20 January 2014

NCIH1975 : Mutation Detection[edit]

Padlock Probes[edit]

  • 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[edit]

  • Experiment Plan
    1. Anneal Padlock probes – 4 probes
      • EGFR T790M(Wild, Mutant), EGFR L858R(Wild, Mutant)
      • 50nM each in 2x SSC + 30% Formamide (2uL each)
      • Preheat probes at 80C for 5min, incubate at 50C for 4hr.
    2. Add SNL mix (Ampligase + KlenTaq enzyme mix)
    3. Incubate 50C for 20hr.
    4. Anneal Primers for 2nd RCA
    5. Run RCA reaction for overnight(20hr.)
    6. Anneal dcProbes, and Image


File:ReactionMix Ampligase KlenTaq.png