Haynes Lab:Notebook/CRISPR Editing/2015/06/30: Difference between revisions
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:aspirate media | :aspirate media | ||
:add | :add 400ul PBS, rock plate, aspirate PBS | ||
:add | :add 200ul trypsin, let sit for a few minutes, tap plate to unstick cells | ||
:add | :add 500ul media, pipette to collect cells, move to 1.5mL microcentrifuge tube | ||
:remove tubes from hood | :remove tubes from hood | ||
:spin in benchtop centrifuge at 0.2x1000RCF for 3 minutes | :spin in benchtop centrifuge at 0.2x1000RCF for 3 minutes | ||
:remove media with P1000 | :remove media with P1000 | ||
:resuspend in 400ul cold PBS | :resuspend in 400ul cold PBS | ||
: | :filter 200ul for flow, use remaining 200ul for gDNA extraction | ||
<br> | <br><br><br> | ||
'''Plating cells for transfection with siRNAs with oligofectamine'''<br> | |||
''oligofectamine calls for no antibiotics, serum-free media, and for cells to be 30-50% confluent at time of transfection'' | |||
:trypsinized Gal4-EED cells that had been induced with dox at 3PM on 6/26 | |||
:filtered some DMEM only for serum-free media | |||
:Plated 0.1x10^6 cells per well of a 6-well plate | |||
Revision as of 10:15, 1 July 2015
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06/30/2015transfected Luc14s gal4-eeds with dox with 2 dilutions of g034 in triplicate.
Step 6: add 100ul to each well
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