Hosuk:LabNotes/2013-9-30
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1st Rolonies and ACTB Gene detection in 1st Rolonies together[edit]
Goal[edit]
- Count ACTB Gene out of 1st rolonies
- Analyze how many ACTBs in all cDNA Rolonies quantitatively
Procedure[edit]
- strip previous proves,
- rehybridize Cy5 labeled probe for 1st rolonies, and ATTO 488 labeled probe for ACTB target
- 1uL of 200uM ATTO488-ACTBTargetOn1stRolony + 2uL of 100uM Cy5-1stRolony in 197uL 2x SSC
- Pre-heat probe mix at 60C for 5min, and add the probe mix to two dishes - 0623 3uL Phi29(used ACTB, RAB7A Padlock probing and created 2nd Rolony), and 0623 10uL Phi29 (only 1st Rolonies)
- incubate at RT for 10min, and wash with 2x SSC twice, store in PBS, and image with Confocal
Result[edit]
- Confocal setting : 20x oil lens --> 580um x 580um view area (1x zoom)
- 512 x 512 pixel seemed not enough, 1024 x 1024 or 2048 x 2048 would be good for recognizing rolonies
- 1 pixel size of 512 resolution was 1.14um which is slightly bigger than normal size of rolony (~700nm)
- 1024 res. --> 1 px = 580um, 2048 res. --> 290um for 1 pixel
- I've taken tile scan images --> 8 x 6 tiles, with 19 stacks along z axis. --> Total scan are = 4.64mm x 3.48mm
- For tile scan, we used 512x512 resolution, and the total scan took about 35 min.
- However, we're going to use 2048 resolution for the next time.
- Convert max. projection for image analysis
- Analyzing images is on going.
- About 30 cells in one field of view (580um x 580um, 512x512 pixel resolution with 20x obj.) --> Total number of cells = ~ 1440
- But by the temporary analysis,
- Roughly, the ratio (total rolonies)/(ACTB target rolonies) in 8x6 tiles = ~ 4900/140000 = ~ 3.5%
PGP1F in 96well plate[edit]
- For testing multiple sets, cells are split in glass bottom 96 well plate.
- 2 well plates are used, and 8 wells in one plate have cells.
Split condition[edit]
- About 90% confluenced cells in T25 flask
- After spin dow cells, add 2mL media,
- transfer 60uL cells in a well, and add 100uL media
- Let's see how long it takes time for 100% cells in a well.