Efficient transfection of Free style 293 cells using Targefect-293F
An easy more economical solution for obtaining high levels of protein expression.
Targefect-293F is a liposomal transfection reagent specially formulated for efficient transfection of free style 293 cells and suspension CHO cell cultures.
Transfection Protocol
The Targefect-293FS reagent should be kept frozen at -20 o C. Thaw the reagent to room temperature just before use. Vortex the reagent at full speed for 30 seconds 2-3 times just before use.
When 293S cells have grown to a high enough density (2 million cells/ml in 50ml or more) proceed to preparing them for transient transfection.
- Retrieve two 125ml Corning shaker flasks.
- Add enough cells and media to get a volume of 27ml at density of 1.1 million cells/ml. Use Freestyle 293 media from Invitrogen or Transfx-293 media from Hyclone.
- Place flasks in shaker incubator at 125rpm and 8% CO2.
- Retrieve GFP DNA and Targefect-293FS Reagent. Thaw both components on ice. Be sure to vortex the lipid vigorously for 30-60 seconds.
- Retrieve two 15ml conical tubes. In each tube add 1.5ml of OPTI-MEM media from Invitrogen. Label one as DNA and the other as Lipid.
- In the DNA tube, add 30ug of GFP DNA and mix by flicking the bottom of the tube. If you are uncertain about the sterility of the DNA you may filter it through a 0.22-micron filter using a syringe and needle.
- In the Lipid tube, add 30ul of Targefect-293FS Reagent and vortex the mixture vigorously for 30-60 seconds.
- Let the DNA and Lipid mixtures incubate for 5 minutes at room temperature.
- At the end of the incubation add the DNA mixture to the Lipid Mixture and mix the contents my flicking the bottom of the tube.
- Let the DNA-Lipid complex's form for 20 minutes at room temperature.
- At the end of 20 minutes add the ~3ml of the DNA-Lipid mixture to one of the two 125ml shake flasks. Label as GFP Control. To the other shake flask add 3ml of OPTI-MEM and label as Cell Only Control.
- Final density in each flask should be ~1x10^6 cells/ml.
- After ~48 hours a green fluorescence should be apparent in cells in the GFP Control flask. After 5 days there should be a very clear difference between the two flasks.
- Harvest both flasks at the end of 5 days. Spin down the cells and save the sup. Store at 4 o C until needed.
Using the above transfection reagent transfection efficiencies greater than 90% have been achieved (using GFP as a marker). Please contact tech support for more information at 1-866-620-4018
Note : For transfecting aderent HEK-293 cells we recoomend the Targefect-293 reagent
Ordering Information
• Targefect-293F, catalog #293F-01, 1ml -------------------------------------- $170
• Targefect=293F, catalog #293F-10, 10 ml --------------------------------- $1250
1453 N Cuyamaca St, El Cajon 92020
Phone: (619)-562-1518, 1-866-620-4018
Fax: (619)-562-1326