Hosuk:LabNotes/2014-1-19
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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
- 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.
- Add SNL mix (Ampligase + KlenTaq enzyme mix)
- Incubate 50C for 20hr.
- Anneal Primers for 2nd RCA
- Run RCA reaction for overnight(20hr.)
- Anneal dcProbes, and Image
- Anneal Padlock probes – 4 probes