Hosuk:LabNotes/2013-6-11
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Day 2
Image processing
- Imaris
- Huygens
Rolony - Day 2
- Cell preparation
- 100% confluence, and coudl wait one more day, and split
- When cells are transferred from 6 well plate
- 500uL of Trypsin, incubate 5min
- Add 500uL media, and mix well, and
- 100uL media in 96 well, or 300~350uL media in glass bottom dish
- Add 60uL of cell in 96 well or 200uL in glass bottom dish
- Culture one or two more day
- BS(PEG)9
- 20uL in 1mL PBS
- Add 200uL of BS(PEG)9 mix per one glass bottome dish
- Add 50uL per one well of 96 well plate
- CircLigase II
- Use 2uL of enzyme, but make 200uL mix by doubling all reagents
- 128uL H2O
- 20uL 10x Buffer
- 10uL MnCl2
- 40uL Betaine
- 2uL CircLigase II
- RCA
- 1~2hr. for RT or 2hr. for CircLigase II would be enough, but
- incubate over 12 hour