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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 <
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