Hosuk:LabNotes/2013-4-22

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Revision as of 21:47, 22 April 2013 by >Hosuki78
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Next Plan

  • Run RCA reaction in a tube with CircLigase II reaction sample using custom DNA
  • Since the custom DNA has the sequence for binding to RCA primer,
  • I could see the signal from RCA product.


  • Experiment
    • Run CircLigase II with custom ssDNA (2hr. @ 60C)
    • Run Exo I (1hr. @ 37C)
    • Tube1 : custom ssDNA + RCA (18hr. @ 30C)
    • Tube2 : Circ + RCA (18hr. @ 30C)
    • Tube3 : Circ and Exo I + RCA (18hr. @ 30C)


  • (As following Jay's suggestion, 18hour RCA was done from yesterday to today using fixed cells, but couldn't get any reasonable result. There were some FL signal, but it was false signal)
    • Jay's suggestion
      • for now, just wash the cells well before you fix. (2 ml PBS x 3)
      • after fixing cells and treating with etoh, wash with PBS.
      • then just dilute your DNA in 200 ul of 2x SSC and incubate at RT for 15 min.
      • wash with 2xSSC several times.
      • then just do your RCA. (with aminoallyl dUTP), followed by the BS(PEG)9 step. then hybridize. You should get a lot of rolonies.