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*[[Hosuk:Lab_Notes|LabNote]] ===1st Rolonies and ACTB Gene detection in 1st Rolonies together=== ====Goal==== *Count ACTB Gene out of 1st rolonies *Analyze how many ACTBs in all cDNA Rolonies quantitatively ====Procedure==== *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==== *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=== *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==== *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.
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