AAT Bioquest

Do you have a suggested protocol for cell transfection?

Posted February 19, 2020


Your optimal protocol will depend on multiple factors including your chosen cell line, but we do have a suggested protocol for our transfection reagent.

Sample Protocol below or available here:

1.  Prepare cells for transfection

  • Culture cells to ~90% confluency and use fresh, clean growth medium

2.  Prepare Transfectamine™ 5000-DNA mixture

  • First mix 2.5 ug of DNA with 200 uL of serum-free medium.
  • Add 7.5 uL of thawed Transfectamine™ 5000 to DNA/medium mixture.
  • Mix well and incubate at room temperature for 20 minutes. 

3.  Add Transfectamine™ 5000-DNA mixture to cell culture

4.  Culture overnight

5.  Analyze transfection efficiency with appropriate method 

  • Maximum expression levels can be discerned 72-96 hours post-transfection
  • If using Flow Cytometry, Laser-Scanning Molecular Imaging, Microscopy, or Western Blot Analysis, a fluorescent label is necessary or strongly recommended




Additional resources

Transfectamine™ 5000 Transfection Reagent