Hosuk:LabNotes/2013-6-11: Difference between revisions
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>Hosuki78 (Created page with "*LabNote ====Day 2==== =====Image processing===== *'''Imaris''' *'''Huygens''' =====Rolony - Day 2===== *BS(PEG)9 **20uL in 1mL PBS *CircLigase II") |
>Hosuki78 No edit summary |
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=====Rolony - Day 2===== | =====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 | *BS(PEG)9 | ||
**20uL in 1mL PBS | **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 | *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 |
Revision as of 15:49, 11 June 2013
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