Hosuk:LabNotes/2014-1-27

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NCIH1975 : Mutation Detection


2nd Rolony

  • Annealing Padlock probes, Ampligase, and 2nd RCA fab. --> done at 01/19~01/21


Detection of Mutation

  • Procedure
    1. Add Cy5-Adapt to detect 1st Rolonies, 3 positions
    2. Strip (80% Formamide for 15min), Add dcProbe1 (incubate 15min), image
    3. Strip (80% Formamide for 15min), Add dcProbe2 (incubate 15min), image
    4. Strip (80% Formamide for 15min), Add dcProbe3 (incubate 15min), image
    5. Strip (80% Formamide for 15min), Add dcProbe4 (incubate 15min), image


Result

  • Image at 3 different positions


1st Rolony
  • Pos1 : 5151
  • Pos2 : 5964
  • Pos3 : 6485

File:NCIH1975 Mutant Pos1-2-3 1stRolony.png


Each dcPobes
  • Not many, just a few, 10~20 from 2nd rolonies.
  • Need positive control or some information about expected abundance of each gene.

File:NCIH1975 Mutant dcProbeCount.png


  • Images of each gene at each position
    • Circles are the 2nd Rolony that I've counted.


File:NCIH1975 Mutant Pos1 dcProbe.png


File:NCIH1975 Mutant Pos2 dcProbe.png


File:NCIH1975 Mutant Pos3 dcProbe.png