Haynes Lab:Notebook/Engineering PC-TFs/2015/02/11

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Summary

Seeding GAL4EED-Luc Cells
GAL4EED Luc cells looked about 90% confluent.
(8.4 x 10^6 cells) / (10mL trypsin and media) = 0.84 x 10^6 cells/mL trypsin and media
(0.84 x 10^6 cells/mL) x Yml = 0.2 x 10^6 cells desired
X = 0.238 mL/well
6 wells --> 1.428mL cells/media/trypsin per 6 well plate. Seeded two plates (2.456mL total)

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.

Remainder used to passage into new T-75. Split 1:10.

New GAL4EED-Luc media made. 10% FBS, 1% P/S, 0.5μg/mL puro.

Transformation
Started re-transformation of successfully constructed CMV-BL09_MV9 and CMV-BL01_MV9.
Used 50μL DH5α-T cells, 10μL dH2O, and 1μL plasmid. Incubated on ice for 5 minutes. Plated on warmed LB Amp plates and incubated at 37°C overnight.