Hosuk:LabNotes/2013-6-11: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>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
Line 8: Line 8:


=====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