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Uses and Applications of FuGENE 6 Transfection Reagent

Protocol Instructions for Users Who are New to Transfection

Below, a standard protocol for the DNA transfection of cultured cells using FuGENE 6 is presented that has been specially designed for scientists who are new to transfection.

1. Prepare cells the day before transfection
  • Ensure the cells are growing well and are in log phase. Occasionally cells slow down when they reach confluence, and it can take a day or two to recover.
  • For monolayers, plate the cells so they will only be 50-80% confluent the following day.

  • Plate the cells in medium that does not contain antibiotics. Transfection is lower in some cell lines when they are grown in antibiotics. Possible explanations are that the transfection reagent may bring the antibiotic into the cell and decrease transfection, or the complex may interact with the antibiotic, resulting in less transfection.
  • For most adherent cell lines, plate 100,000-300,000 cells in 2 ml medium (50,000 to 150,000 cells/ml) in one well of a 6-well plate. Plan to test at least 3 ratios of reagent to DNA.. See the table below for a suggested layout of a 6-well plate.
(A) Cell Control

    No reagent

    No DNA
(B) Reagent Control

    6 l reagent

    No DNA
(C) DNA-Control

    No reagent

    1 g DNA
(D) 3:1 ratio

    3 l reagent

    1 g DNA
(E) 3:2 ratio < p>
    3 l reagent

    2 g DNA
(F) 6:1 ratio

    6 l reagent

    1 g DNA

2. Prepare the complex example given for 6-well plate

Prepare the DNA

  • Select a plasmid that you know transfects your cell line, and make sure you are able to measure the expression of reporter gene or transfected protein. Plasmids with reporter genes are available from Roche Applied Science.
  • Handle the DNA as aseptically as possible to avoid contaminating your cell cultures during the transfection. Use sterile pipettes, tips, vials, etc.
  • Accurate DNA concentrations are important. Determine the concentration of the DNA with a spectrophotometer. Transfection-grade DNA should have a 260/280 ratio of 1.8.
  • Check the stock DNA concentration, and determine how much you will need for 1 and 2 g. If the stock is 2 g/l, then decide if you can accurately pipet 0.5 l. It might be better to dilute the DNA using procedures that give accurate dilutions. If you need to dilute the DNA, use sterile DNase-free water or sterile DNase-free TE buffer as the diluent.
  • Calculate accordingly for your DNA concentrations (i.e., concentrations of 0.5 g/l work well; for 1 g, measure 2 l, and for 2 g, measure 4 l).
Prepare the FuGENE 6 Transfection Reagent

  • Remove the FuGENE 6 Reagent from the refrigerator or freezer and allow it to warm to room temperature. Meanwhile, look at the cells microscopically. Make sure they look healthy and are 50-80% confluent. Return the cells to the incubator.
  • Warm serum-free DMEM (no additives) to room temperature. Aliquot 94 -100 l into each of 6 tubes as indicated below. Use sterile Eppendorf tubes or small sterile polystyrene tubes labeled:
    • A (100 l)
    • B (94 l)
    • C (100 l)
    • D (97 l)
    • E (97 l)
    • F (94 l), corresponding to the plate layout above.
  • Mix the room-temperature FuGENE 6 Reagent by tapping or briefly vortexing (1 second).
    Carefully add the FuGENE 6 Reagent to the serum-free medium as follows.
    • 6 l to Tube B
    • 3 l to Tube D (3:1 ratio)
    • 3 l to Tube E (3:2 ratio)
    • 6 l to Tube F (6:1 ratio)
  • Ensure the entire amount of reagent is delivered directly from the pipette tip into the serum-free medium. Avoid contact of the FuGENE 6 Transfection Reagent with the walls of the tube containing the serum-free medium. Avoid a FuGENE 6 Reagent layer on top of the serum-free medium. Once the FuGENE 6 Reagent is added, immediately tap, flick, or vortex for 1 second to ensure adequate mixing of the components.
  • Incubate at room temperature for 5 minutes.

Make the complex

Add the DNA to each tube (see table below). The amount added will depend on the starting concentration of DNA.

Tube DNA at 0.5 g/l DNA at 1 g/l DNA at 2 g/l
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