Hosuk:LabNotes/2013-7-1: Difference between revisions

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>Hosuki78
(Created page with "*LabNote ===Rolony, 12th : 96 well plate=== *Check point *#Epicenter vs. Enzymatics *#1uL vs. 3uL Phi29 *#Brown vial vs. white vial a-dUTP of Anaspec *#5u...")
 
>Hosuki78
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*16 well experiment set
*16 well experiment set
[[File:ExpSet_96well.png|500px]]
[[File:ExpSet_96well.png|300px]]




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**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.”
**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?
**I don't know what's the problem in white vial, a-dUTP might be degraded?
*Enzymatics phi29 looked working better slightly than Epicentre one, but not 100% sure.
*Enzymatics and Epicentre Phi29 don't have significant difference.
**1uL of Enzymatrics and 3uL of Epicentre!
**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+Rnase H = 2+10 and 5+5 are working well, there are no difference.
**Riboshredder : 1U/uL, 100uL or 500uL stock, and
**Riboshredder : 1U/uL, 100uL or 500uL stock, and
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**2uL + 10uL -->  1unit + 1unit at each
**2uL + 10uL -->  1unit + 1unit at each
*3uL Phi29 seemed generating rolonies a little more, but 1uL would be enough!
*3uL Phi29 seemed generating rolonies a little more, but 1uL would be enough!




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====Montage of Rolony Images at each conditions====
====Montage of Rolony Images at each conditions====
*Raw images : [[Media:Montage3_Stack1_middlesize.tif|Montage]]
*Raw images : [[Media:Montage3_Stack1_middlesize.tif|Montage]]
[[File:Montage2_Stack1_EditCaption.png|500px]]
[[File:Montage2_Stack1_EditCaption.png|800px]]

Latest revision as of 21:24, 2 July 2013

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