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Uses and Applications of FuGENE 6 Transfection Reagent

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    4 l
    2 l
    1 l
    F
    2 l
    1 l
    0.5 l
  • Mix by tapping or vortexing the tube for 1 second.
  • Incubate for at least 15 minutes at room temperature.
  • 15 minutes is the minimum incubation time. Longer complex incubation times of 30-60 minutes may enhance transfection efficacy; however, this is cell-line dependent. If you want to test this, you can prepare either a separate tube for each transfection reaction or a master mix, then pull aliquots at each time point. For example, to make a master mix for the 3:1 ratio:
    • Add 30 l FuGENE 6 Reagent to 970 l serum-free medium. Mix, then incubate 5 minutes.
    • Add 10 g DNA to the diluted FuGENE 6 Reagent. Mix, incubate for 15 minutes, then start adding 100-l aliquots to cells at 15-minute intervals.
    • A slight haze may be visible if the transfection complex is in clear tubes.

3. Add the complex to the cells
  • Remove the cells from the incubator.
  • Add 100 l of complex drop-wise to its designated well as in the plate outline above. Swirl the plate to ensure mixing of the complex in all of the wells.
  • Return the cells to the incubator.

4. Incubate the cells until the time of assay
Incubate the cells for 1-3 days prior to measuring gene expression. The incubation period is dependent on the reporter gene in the plasmid. If the plasmid contains a selectable marker, do not add the selection antibiotic until the first post-transfection passage.
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