Hosuk:LabNotes/2013-6-11

From ZhangLabWiki
Jump to navigation Jump to search

Day 2[edit]

Image processing tools[edit]
  • Really powerful and necessary tools, expensive though
  • Imaris
    • for rendering, image processing
  • Huygens
    • for deconvolution and image alinging


Image processing procedure[edit]
    • Take raw images data to storage server. Raw images from Confocal is 12 bit
    • Open raw images with Imaris, and export to Huygens
    • Deconvolution with Huygens, end export back to Imaris (become 32 bit)
    • Check images with Imaris, and reduce image size
      • Maximum projection --> 2D image from 3D z stack images
      • Downsampling to 8 bit
      • Reduce resolution from 4000 x 4000 to 1000 x 1000
    • Link time series
    • Create mask for aligning with Imaris and align images with Huygens
    • Convert time to z --> Get one image has pixel towers (stacks) of each time series of each individual rolony signal
    • Use genome libraries to match each colors of rolonies to known libraries, without concerning further image processing to remove noise, background and resolve which pixel is real or not.


Rolony - Day 2[edit]
  • Cell preparation
    • 100% confluence, and could 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


  • After CircLigase reaction (for 2hr.)
    • Use PBS for washing 3 times


  • RCA
    • 1~2hr. for RT or 2hr. for CircLigase II would be enough, but
    • incubate over 12 hour


  • Cy3 probe hybridization (using Jonathan's sample)
    • 1 uL of 100um probe in 1 mL 2x SSC
    • and add 200uL in glass bottom dish, 50uL for 96 well
    • Add Cy3 probe, incubate at RT for 15min
    • Wash once with 2x SSC
    • Wash once with 1x SSC
    • Wash with PBS, store in PBS