转染步骤
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6272
Procedure for Transfection of Mammalian Cells
Materials:
Lipofectamine
IMDM containing 10% fetal bovine serum, 1% glutamine, 1% aa
IMDM containing 1% glutamine
IMDM containing 20% fetal bovine serum, 1% glutamine, 1% aa
(1)In a six-well or 35 mm tissue culture plate, seed ~2x 105 cells per well in 2 ml IMDM containing 10% FBS and nonessential amino acids.
(2)Incubate the cells at 37E C in a CO2 incubator until the cells are 70-80% confluent. This will usually take 18-24 h.
Prepare the following solutions in 12 x 75 mm sterile tubes:
(3)Solution A: For each transfection, dilute 2 μg DNA (plasmid) in 375 μl serum-free IMDM (containing nonessential amino acids).
(4)Solution B: For each transfection, dilute 12 μl LIPOFECTAMINE Reagent in 375 μl serum-free IMDM.
(5)Combine the two solutions, mix gently, and incubate at room temperature for 15-45 min. The solution may appear cloudy, however this will not impede the transfection.
(6)Wash the cells once with 2 ml serum-free IMDM.
(7)For each transfection, add 750 μl serum-free IMDM to each tube containing the lipid-DNA complexes. Do not add antibacterial agents to media during transfection. Mix gently and overlay the diluted complex solution onto the washed cells.
(8)Incubate the cells for 5 h at 37E C in a CO2 incubator.
(9)Add 1.5 ml IMDM with 20% FBS without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium.
(10)Replace medium at 18-24 h following start of transfection.
(11)Assay cell extracts for gene activity 24-72 h after the start of transfection, depending on cell type and promoter activity.
Materials:
Lipofectamine
IMDM containing 10% fetal bovine serum, 1% glutamine, 1% aa
IMDM containing 1% glutamine
IMDM containing 20% fetal bovine serum, 1% glutamine, 1% aa
(1)In a six-well or 35 mm tissue culture plate, seed ~2x 105 cells per well in 2 ml IMDM containing 10% FBS and nonessential amino acids.
(2)Incubate the cells at 37E C in a CO2 incubator until the cells are 70-80% confluent. This will usually take 18-24 h.
Prepare the following solutions in 12 x 75 mm sterile tubes:
(3)Solution A: For each transfection, dilute 2 μg DNA (plasmid) in 375 μl serum-free IMDM (containing nonessential amino acids).
(4)Solution B: For each transfection, dilute 12 μl LIPOFECTAMINE Reagent in 375 μl serum-free IMDM.
(5)Combine the two solutions, mix gently, and incubate at room temperature for 15-45 min. The solution may appear cloudy, however this will not impede the transfection.
(6)Wash the cells once with 2 ml serum-free IMDM.
(7)For each transfection, add 750 μl serum-free IMDM to each tube containing the lipid-DNA complexes. Do not add antibacterial agents to media during transfection. Mix gently and overlay the diluted complex solution onto the washed cells.
(8)Incubate the cells for 5 h at 37E C in a CO2 incubator.
(9)Add 1.5 ml IMDM with 20% FBS without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium.
(10)Replace medium at 18-24 h following start of transfection.
(11)Assay cell extracts for gene activity 24-72 h after the start of transfection, depending on cell type and promoter activity.
