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

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====Day 2====
====Day 2====
=====Image processing=====
=====Image processing tools=====
*Really powerful and necessary tools, expensive though
*'''Imaris'''
*'''Imaris'''
**for rendering, image processing
*'''Huygens'''
*'''Huygens'''
**for deconvolution and image alinging
=====Image processing procedure=====
**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=====
=====Rolony - Day 2=====
*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
*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
*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

Latest revision as of 21:16, 11 June 2013

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