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

Specific technique considerations

Co-transfections

First, verify that both of your DNA constructs work in your cell line. Also, determine the ratio of DNA to FuGENE 6 Reagent (for example, 1 g of DNA and 3 l FuGENE 6 Reagent) that provides good expression levels. Then, perform the transfection, ensuring the DNA / FuGENE 6 Reagent ratio takes into account the total amount of DNA from the two constructs. For example, in the above experiment the co-transfection of 1 g each of two carefully measured DNA constructs would require 6 l of FuGENE 6 Transfection Reagent (2 g DNA to 6 l of FuGENE 6 Transfection Reagent). FuGENE 6 Transfection Reagent is very gentle, so there will not be a cytotoxic effect when an excess of FuGENE 6 Transfection Reagent is used.

Using spinner flasks

The special components (e.g., Pluronic F-68) that are added to the spinner flask media for cell protection can interfere with transfection. To overcome this problem:

At the time of subculturing, resuspend the cells in medium that does not contain protective reagents, then perform the transfection in a flask or Petri dish for one hour as suggested in the package insert. For example, for 1 g DNA, use 3 l FuGENE 6 Reagent for each 100,000-200,000 cells. The cell concentration may be several times the concentration you will use for seeding. After allowing an hour for transfection, dilute into your normal suspension medium, then place it into the spinner flask.

Producing stable transfectants

Do not use a large number of plates, or plate fewer cells per plate. FuGENE 6 Reagent is so gentle and efficient that you will obtain many transfected cells. Use a good construct and good selective medium, and you will obtain a large number of clones.

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