Hosuk:LabNotes/2013-7-1

From ZhangLabWiki
Jump to navigation Jump to search

Rolony, 12th : 96 well plate[edit]

  • Check point
    1. Epicenter vs. Enzymatics
    2. 1uL vs. 3uL Phi29
    3. Brown vial vs. white vial a-dUTP of Anaspec
    4. 5uL+5uL vs. 2uL+10uL of Riboshredder + Rnase H mix


  • Start at 06/29
    • Use cell dishes fixed (10% Formalin once) at 06/26 in 16 well of 96 well plate
    • Run RT : 6:00pm 06/29 ~ 6:00am 06/30 --> ~12hr.
    • Run CircLigase II : 9:00am 06/30 ~ 1:20pm 06/30 --> ~4hr.
    • Run RCA : 2:50 pm 06/30 ~ 10: 50am 07/01 --> ~20hr.


  • 16 well experiment set

File:ExpSet 96well.png


Result[edit]

  • Brown a-dUTP does work while white vial does not.
    • Service of AnaSpec replied that “According to our production team,  there are no difference between the two. Both products are in TE buffer with a concentration of 4 mM.”
    • I don't know what's the problem in white vial, a-dUTP might be degraded?
  • Enzymatics and Epicentre Phi29 don't have significant difference.
    • 1uL is good for Enzymatrics and 3uL for Epicentre!
  • Riboshredder+Rnase H = 2+10 and 5+5 are working well, there are no difference.
    • Riboshredder : 1U/uL, 100uL or 500uL stock, and
    • Rnase H : 5u/uL, 1mL stock
    • 2uL + 10uL --> 1unit + 1unit at each
  • 3uL Phi29 seemed generating rolonies a little more, but 1uL would be enough!


In final protocol[edit]

  • Brown vial of animoallyl dUTP
  • 2uL + 10uL of Riboshredder + RNase H
  • 1uL of Enzymatrics or 3uL of Epicentre
  • 4hr. CircLigase II


Montage of Rolony Images at each conditions[edit]

File:Montage2 Stack1 EditCaption.png