Haynes Lab:Notebook/Jan/2015/09/04: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==Isolating Transfected U2OS BD004/BD006 Colonies== | ||
* | * With the BD006 1:10 plate and BD004 1:10 plate: | ||
** Split 1:10 into another 10 cm plate and | |||
** Split 1:10 into a second 10 cm plate (in effect, a 1:100 split). | |||
* These plates will be used to determine if we can separate positive from negative cells by FACS. | |||
* With both 1:20 plates: | |||
** Removed old media and added standard media with 0.5 ug/mL puromycin to see if this is enough to kill off negative cells. | |||
* With 1:40 plates: | |||
** Made a grid on a piece of tape and stuck it to the bottom of the 10 cm plate. | |||
** Used fluorescent microscope and grid to write down approximate location of groups of fluorescent cells. | |||
** Acquired 17 locations for both plates; set up TC hood. | |||
** Took out one plate at a time, washed with 5 mL of PBS, then added '''5 mL''' of trypsin and let sit in the hood at RT for 5 min '''without moving the plate at all'''. | |||
** During the 5 minutes, placed 900 uL of standard U2OS medium in 17 wells in a 24-well plate. | |||
** After the 5 minutes were over, took 50 uL of trypsin and cells from the location written down '''without moving the plate''', then took another 50 uL from the same location. | |||
** Repeated this for the other 16 wells and the other plate. | |||
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Latest revision as of 01:08, 27 September 2017
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Isolating Transfected U2OS BD004/BD006 Colonies
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