Haynes Lab:Notebook/Short Projects/2014/09/10: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Haynes Lab - Short Projects</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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==09/10/14 - Jonah Thomas== | ==09/10/14 - Jonah Thomas== | ||
* Cell culture - Gal4-EED/luc for luciferase activation experiment | * Cell culture - Gal4-EED/luc for luciferase activation experiment | ||
* Passage cells 1: | * Passage cells 1:4 (T-75 flasks) | ||
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'''Cell culture''' | '''Cell culture''' | ||
* Make fresh growth medium: DMEM, 10% FBS, 1% pen-strep | |||
* Set up 2x 6-well plates for Gal4-VP64 transfections | * Set up 2x 6-well plates for Gal4-VP64 transfections | ||
** 6 wells dox- (3 will be transfected, 3 not) | ** 6 wells dox- (3 will be transfected, 3 not) --> 2.5E5/well | ||
** 6 wells dox+ (3 will be transfected, 3 not) | ** 6 wells dox+ (3 will be transfected, 3 not) --> 2.5E5/well | ||
# Harvest cells: Follow steps 1-9 of [http://openwetware.org/wiki/Haynes:SplittingCells Passaging Adherent Cells] protocol.<br>Note: cells are ~75% confluent, so at this point you will have ~7.5E5/ mL = ~7.5E6 in 10 mL. | |||
# Calculation 2: How much growth medium used per unique plate? 6 (+1) wells x 4 mL = 28 mL growth medium | |||
# Calculation 3: How many cells used per unique plate? 6 (+) wells x 2.5E5 cells = 1.75E6 cells | |||
# Calculation 4: How much stock volume of harvested cells = Calc 3? 1.75E6 cells / (~7.5E5/ mL) = 2.3 mL | |||
# In a 50 mL conical, add '''25.7 growth medium''' (which is 28 - 2.3) | |||
# Add '''2.3 mL of harvested cells''' to the 50 mL conical. | |||
# Get a 10 mL pipette and mix the cells by pipetting up and down (>3 times). | |||
# Draw up and dispense 4 mL of the suspended cells into each well in the 6-well plate. | |||
* Also, passage cells 1: | Note: Do this whole procedure separately for the dox- and the dox+ cells | ||
* Also, passage cells 1:4 in T-75 flasks | |||
# Get a new T-75 | |||
# Add 7.5 mL of growth medium to the flask | |||
# Add 2.5 mL of harvested cells to the flask | |||
Latest revision as of 00:16, 27 September 2017
Haynes Lab - Short Projects | Main project page Next entry |
09/10/14 - Jonah Thomas
Cell culture
Note: Do this whole procedure separately for the dox- and the dox+ cells
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