Hosuk:LabNotes/2013-6-11
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