Hosuk:LabNotes/2013-7-1
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Rolony, 12th : 96 well plate[edit]
- Check point
- Epicenter vs. Enzymatics
- 1uL vs. 3uL Phi29
- Brown vial vs. white vial a-dUTP of Anaspec
- 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
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]
- Raw images : Montage