Haynes Lab:Notebook/Engineering PC-TFs/2015/02/11: Difference between revisions
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==Summary== | ==Summary== | ||
'''Seeding GAL4EED-Luc Cells'''<br> | |||
GAL4EED Luc cells looked about 90% confluent.<br> | |||
(8.4 x 10^6 cells) / (10mL trypsin and media) = 0.84 x 10^6 cells/mL trypsin and media<br> | |||
(0.84 x 10^6 cells/mL) x Yml = 0.2 x 10^6 cells desired <br> | |||
X = 0.238 mL/well<br> | |||
6 wells --> 1.428mL cells/media/trypsin per 6 well plate. Seeded two plates (2.456mL total) <br> | |||
<br> | |||
Added 3.5mL additional GAL4EED-Luc media to each well to bring total volume to 4mL. Added 4μL 1mg/ml doxycycline to top three wells and marked as such to bring to 1μg/ml.<br><br> | |||
Remainder used to passage into new T-75. Split 1:10.<br><br> | |||
New GAL4EED-Luc media made. 10% FBS, 1% P/S, 0.5μg/mL puro.<br><br> | |||
'''Transformation'''<br> | |||
Started re-transformation of successfully constructed CMV-BL09_MV9 and CMV-BL01_MV9.<br> | |||
Used 50μL DH5α-T cells, 10μL dH<sub>2</sub>O, and 1μL plasmid. Incubated on ice for 5 minutes. Plated on warmed LB Amp plates and incubated at 37°C overnight. | |||
Revision as of 11:00, 12 February 2015
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SummarySeeding GAL4EED-Luc Cells Transformation
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