Hosuk:LabNotes/2013-4-24

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RCA with Pre-circularized DNA on fixed cells[edit]

Procedure[edit]
  • Run 18hr. RCA with using CircLigase II product got from 4/24 result
    • 7uL of product + 93uL 2x SSC in the fixed cell dish
    • incubate 15min at RT
    • Wash with PBS once
    • Run RCA with the same step as rolony protocol
      • 1uL of 100uM RCA primer in 199uL 2x SSC
      • Add to cell, incubate for 15min at 60C
      • Aspirate, and wash using 2x SSC twice
      • Aspirate, and wash using 0.1x SSC twice
      • Add RCA mix
    • 18hr in 30C Oven


Result[edit]
  • FL spots looked similar number and similar density to what I’ve got before (e.g. 03/05).
  • Those seemed rolony but not many.
  • It is suspected that rolonies didn't bind inside cell so they could be washed away.
  • Are there problem in washing step? or BS(PEG)9 procedure?
  • Need to ask to Jay


20x Obj. Bright field 20x Obj. Cy3 Merged
(Original) (Original)
File:S1 washPBS3times PreCirc RCA18hr Cy3Adapt 20x pos02 fig04 BF Exp0.2-EM00.jpg File:S1 washPBS3times PreCirc RCA18hr Cy3Adapt 20x pos02 fig03 Cy3 Exp0.2-EM40.jpg File:Composite fig03-04.jpg