Hosuk:LabNotes/2013-6-18: Difference between revisions

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*[[Hosuk:Lab_Notes|LabNote]]
*[[Hosuk:Lab_Notes|LabNote]]


====Rolony - 9th====
===Rolony, 9th===
*Try the method that I’ve learned from Jonathan and Jay
*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)
*2uL Riboshredder + 10uL Rnase H + 20uL Rnase H buffer + 168uL H2O (Jonathan’s protocol)

Revision as of 17:31, 21 June 2013

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!
    • Low background, and no signal through FITC nor Cy5 channel though,
    • Use sigma's Betaine because of running out of original betaine in CircLigase II kit, would it be reason? not sure!
  • Need to try one more! using original protocol of CircLigase II!


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