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http://mcardle.oncology.wisc.edu/sugden/Protocols/html files/Establishing stable maxiEBV producing cells (Establishing stable 293_maxiEBV clones).htm
Establishing stable 293/maxiEBV clones
Seeded 2_106 cells on a 10cm plate
After 2 days, transfected 10ug 3227/2089(maxiEBV) using lipofectamine (Invitrogen)
(1) Mix 10ug DNA with 500ul Optimem
(2) Mix 15ul lipofectamine with 500ul Optimem
(3) Wait for 5min
(4) Mix (1) (2) incubate for 20min
(5) Add (4) to cells
24 hrs after transfection, check for GFP expression
(My transfection efficiency was around 20%. )
Trypsinized cells and plated 1x106 cells (? 2 x105 green cells) on a 15cm plate
1x105 cells (? 2 x104 green cells)
1x104 cells (? 2 x103 green cells)
Added 200ug/ml Hygromycin for selection
Waited 2-3 weeks for clones to grow.
My cloning efficiency was around 0.1% (1/103 green cells gave rise to clones).
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