Department of Biological Sciences,
1000 Hilltop Circle
Baltimore, MD 21250
AMB Program Biology Home Page Laboratory Manual AMB Course Page


Procedure for Transfection of Mammalian Cells 


bullet Lipofectamine (Invitrogen)
bullet IMDM containing 10% fetal bovine serum, 1% glutamine, 1% aa
bullet IMDM containing 1% glutamine
bullet 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 37°C in a CO2 incubator until the cells are 70-80% confluent. This will usually take 18-24 h.
  3. Prepare the following solutions in 12 x 75 mm sterile tubes:
Solution A: For each transfection, dilute 2 μg DNA (plasmid) in 375 μl serum-free IMDM (containing nonessential amino acids).
Solution B: For each transfection, dilute 12 μl LIPOFECTAMINE Reagent in 375 μl serum-free IMDM.
  1. 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.Wash the cells once with 2 ml serum-free IMDM.
  2. 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.
  3. Incubate the cells for 5 h at 37°C in a CO2 incubator.
  4. 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.Replace medium at 18-24 h following start of transfection.
  5. Assay cell extracts for gene activity 24-72 h after the start of transfection, depending on cell type and promoter activity.

This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on 3/2/2010

 !   Nine month MS Degree


Master's Degree in Applied Molecular Biology (AMB)

is designed for students interested in careers in industrial and biomedical sciences.

Useful links:

REBASE Homepage
Science Advisory Board

Looking for career advice?

FindHow - Biology Careers