Hosuk:LabNotes/2013-6-10

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Training Day 1[edit]

  • Some of important things
  • Phi29 enzyme from Enzymatics is used.
  • Actually Jay likes to use (or trusts) Enzymatics, so almost all reagents are from Enzymatics.
  • The concentration of Phi29 of Enzymatics that Jonathan has used is 100U/uL which is High concentration one from Enzymatics, and this concentration is the same as low concentration one from Epicentre.
    • 1uL of phi29 in 200uL total mix
    • I could try 3uL of phi29


  • RNase H step after RT is critical step, so do this carefully.
  • RNase H + Riboshredder mix is
    • 10uL of RNase H +
    • 2uL of Riboshredder +
    • 20uL of 10x Buffer +
    • 168uL of H2O


  • Try HeLa cells unless I get rolonies with PGP1F.


  • The sample (that I've got some signal at 05/29) doesn't seem to have actual rolonies,
  • There are no dots, just too much even signal distribution.


  • Jonathan fixed cells just using 10% Formamide
    • Aspirate media
    • Add 50uL of 10% Formamide (200uL for glass bottom)
    • Incubate for 15min
    • Wash with PBS three times
    • Store in Ethanol



Rolony Construction[edit]
  • S1 : +RT, +CircLigase, +RCA
  • S2 : -RT (no RT enzyme), +CircLigase, +RCA
  • Jonathan's samples in 96 well (2 positive, 2 negative controls)
  • Jonathan fixed his cells in 96 well plate, using 10% Formalin and incubating 15min, without 37% Formalin.
  • HCL incubation for 1min.


  • Run RT for overnight (5:00pm)