( ...)Uses and Applications of FuGENE 6 Transfection Reagent

Troubleshooting Hints if You Observe Cytotoxicity (greater than 5 - 10 % Cell Death)

FuGENE 6 Transfection Reagent has proven to be virtually nontoxic to most cell types. If you observe more than 5 - 10 % cell death in transient transfection experiments, or if selection of permanent transfectants fails, refer to the table below for possible causes.

Possible Cause Recommendation Selection antibiotic added too soon

Repeat transfection and wait an additional 24 to 48 h before adding the selection antibiotic to allow for sufficient expression of the selection marker. Selection antibiotic concentration too high

Repeat transfection using a lower concentration of selection antibiotic. Transfected protein is cytotoxic or is produced at excessively high levels Reduced viability or slow growth rates may be the result of high levels of protein expression, as the cells metabolic resources are directed toward the production of the heterologous protein. The expressed protein may also be toxic to the cell at the level expressed. To analyze cytotoxicity, prepare experimental controls as described below.

Prepare extra wells containing:

1. Cells that are not transfected.
2. Cells transfected with DNA alone (i.e., without FuGENE 6 Transfection Reagent)
3. Cells treated with FuGENE 6 Transfection Reagent alone (no DNA added).

Compare cells transfected with the experimental construct to wells that contain these experimental controls. Consider repeating th
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