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") |
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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