Hosuk:LabNotes/2013-6-18

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Revision as of 22:23, 21 June 2013 by >Hosuki78
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Rolony, 9th

  • Try the method that I’ve learned from Jonathan and Jay
  • 2uL Riboshredder + 10uL Rnase H + 20uL Rnase H buffer + 168uL H2O (Jonathan’s protocol)
  • Use double volume of CircLigase reagent except CircLigase II (Jonathan’s protocol)
  • Use Enzymatics’ Phi29 --> 1uL Phi29 (High concentration of Enzymatics) for RCA mix
  • Use plastic bag with wet tissue for long time incubation


  • Start at 06/16
  • Use cell dishes fixed at 06/04
  • S3 --> -RT, S4 --> +RT
  • Run RT : 9:00pm 06/16 ~ 9:00am 06/17 --> 12hr.
  • Run CircLigase II : 12:05am 06/17 ~ 4:05pm 06/17 --> 4hr.
  • Run RCA : 5:00 pm 06/17 ~ 3: 10pm 06/18 --> 22hr.


Result

  • S3, -RT sample didn't show any rolony signal.
  • S4, +RT sample: had not as many rolonies as what I expected, but not bad!
    • Low background, and no signal through FITC nor Cy5 channel though,


epi FL

  • -RT, 20x, NA=0.8

File:S3 -RT 20x-NA0.8 pos02.png


  • +RT, 20x, NA=0.8

File:S4 +RT 20x-NA0.8 pos02.png


  • +RT, 40x Oil, NA=1.3

File:S4 +RT 40xOil-NA1.3 pos03.png