#8
Transfection Procedure:


Preferred: Lipofectamine 2000

Day 1:  change cells to media without antibiotics (Optimem I)
90-95% confluent best

Day 2:  Prepare complexes (for 6-well)
a)      Dilute 4ug DNA in 250ul Optimem I without serum/reduced serum. Mix gently
b)      Mix Lipofect 2000 gently, then dilute 10ul +240ul Optimem I . Mix
gently and incubate 5min RT.
c)      Combine diluted DNA with diluted Lipofectamine 2000. Mix gently,
incubate 20 min RT.
d)      Add the DNA-Lipos coplex to each well. Mix gently by rocking back forth
e)      Incubate at 37C incubator 24h  assay transfection


* For Invivo Gene Shuttle (DOTAP:Chol):
a)      For 4ug DNA final, need 8ul 0.5ug/ul soln prepared as IVGS
instructs: 4ug DNA (~1ul)+3ul D5W, mix.
b)      Mix 1.6ul liposomes + 6.4 D5W  (for a 4nm final)
c)      Drop DNA onto diluted liposomes, mix up down 3X
d)      Add DNA-lipos to well. Mix gently, rocking. Add 2ml media Optimem I.
e)      Incubate 37C incubator 24h  assay tfection.