| Steven Finkbeiner, Departments of Neurology and Physiology, UCSF
http://gweb1.ucsf.edu/labs/finkbeiner/Protocol/protocol_list/Six Well Cell Trans.shtml
This is a method devised by Marc Diamond: Primarily, this method uses Lipofectamine plus Reagent as the vehicle for introducing the
Plasmid DNA into the cell.
Ingredients
Opti-mem (serum free-media)- Gibco brand
Lipofectamine and plus Reagent (come packaged together)- Gibco
293 Cell/Cos Cell Media (kept at 4c) make up ourselves
GFP DNA Plasmid
One’s own DNA Plasmid
Take two polystyrene tubes;
Label tubes #1, and#2
Into Tube #1
100ul Opti-mem
3.2ul Reagent Plus
0.8ug GFP
1ug DNA (your own)
Incubate this mix for 5min
Into tube #2
100ul Opti-mem
2.2ul Lipofectamine
Add tube #2 to tube #1. Incubate for 15min
At the same time aspirate away cell media from the six well dish and replace with 37c warm Opti-mem (800ul per six well dish).
After 15 min add tube 1 and 2 mixture to six well dish which contains 800ul Opti-mem solution. Swish solution around a little.
Leave solution in well for 3 hours. After 3 hours aspirate away Lipofectamine/Reagent plus/Opti-mem Media and replace with 293/Cos Cell Culture Media.
Around 48 hours later Lyse Cells.
Note:
Cell Transfection success may lie with the number of cells you begin with on the plate. With a very successful transfection (~50-60%) I started with ~3.2 million cells/dish.
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