Etchevers:Notebook/STRA6 in eye development/2009/05/18

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Seeding HEK 293T cells for extraction

T75 flask had 23 million cells as per counting. I taught Adeline how to count on clumpy cells that had just been detached by PBS - to avoid in the future.

But with the clumps, today, there were approximately 255 x 104 cells/mL - seeded 50K, 100K and 150K cells per well of 24 well plate. This corresponded respectively to 0.48, 1.92 and 1.44 mL cells in 23.5 mL, 46 mL or 22.56 mL medium (two dishes of 100K cells/dish, one on an expired Corning plastic. This grew just fine.)

Also froze 5x ampoules in liquid nitrogen with 2.5 million cells per ampoule (spaces 3-8 in 5th row, yellow tier).

Transfection (HEK) 40' complex incubated, rather than the 30' recommended.

GFP = 150 ng/uL DNA, use 26.4 uL + 173.6 uL NaCl.

For the PAX6 expression vector, use 142 uL NaCl + 8 uL jetPEI x 4


1 ug EGFP qsp 50 uL per well

Either one x 4 vortex, spin, incubate for 15-30 minutes with 100uL per well.