Refer to the following table if you observe low transfection efficiency.
Nucleic acids of poor quality or insufficient quantity
Verify the amount or quality of nucleic acid:
- Use only high-quality plasmid preparations with no contaminating
nucleic acids that can be sufficiently quantitated in a photometer
- Use DNA at a concentration of 0.022.0 g/l
- Verify that the transfected plasmid construct contains appropriate
promoters and other sequences required for protein expression in the
cell line being transfected
- Perform a control transfection experiment with a commercially
available transfection-grade plasmid preparation (e.g., the -gal
control vectors supplied with the Mammalian Expression Vectors for
Epitope Tagging (Cat. No. 1 814 664).
Note: Endotoxins are reported to be cytotoxic to some very sensitive
cell lines (e.g., Huh-7) and primary cultures. When using FuGENE 6
Transfection Reagent for many common cell types, it may be possible to use
DNA that contains higher endotoxin levels.
Insufficient number of cells were used
Use adherent cells at 5080% confluency.
FuGENE 6 Transfection Reagent was aliquotted
and stored in a new container
Ensure the FuGENE 6 Transfection Reagent is stored in the original
FuGENE 6 Transfection Reagent came into
contact with plastic
Repeat transfection, carefully pipetting FuGENE 6 Transfection Reagent
directly into serum-free medium.
Transfection complex was formed in medium containing serum or other
The FuGENE 6 / plasmid complex can be directly added to cells that are
cultivated in complete medium with FCS and other additives. However,
dilution of FuGENE 6 Tranfection Reagent and the formation of the complex
must be done in serum-free medium without any additives. Check the
original bottle of medium used for complex formation. Repeat the
experiment using a new bottle of medium that does not contain any
additives (e.g., serum, antibiotics, growth enhancers, etc.)
Transfection complex formation time was too short
Let the FuGENE 6 Reagent/DNA complex form at least 15 minutes at room
temperature. In some cell lines the transfection rate increases as the
time to form the complex increases up to two hours.
A suboptimal FuGENE 6 vs. DNA ratio was used
Transfection efficiency is greatly reduced if the amount of DNA gets
too high. Optimize the FuGENE 6 Transfection Reagent:DNA ratio according
to the following procedure. Always use more FuGENE 6 Transfection Reagent
(l) than DNA (g). For example, combine 3 l FuGENE 6 Transfection
Reagent with 12 g DNA for a 35-mm culture dish (6-well plate).
Prepare FuGENE 6:DNA mixtures according to the following table. Do not
allow FuGENE 6 Transfection Reagent to come in contact with the plastic
tube before diluting with serum-free medium.
Label six tubes
Add serum-free medium (l)
Add FuGENE 6 Transfection
Add DNA (g)
Amount of Transfection complex was too low
- Gently tap the tubes. Mix thoroughly, but do not vortex.
- Incubate at room temperature for 15-45 minutes.
- Add each FuGENE 6 Transfection Reagent:DNA mixture to a 35-mm
culture dish or one-well of a 6-well plate. Swirl the plates.
- If you increase the DNA concentration (e.g., in a co-transfection
experiment), proportionally increase the amount of FuGENE 6
- If your cell line is not easily transfected by the above FuGENE 6
Transfection Reagent:DNA ratios, test a wider range of ratios,
including 215 l FuGENE 6 Transfection Reagent per 12 g DNA,
per 35-mm culture dish.
- If no transfection is observed, repeat the experiments with DOTAP or
DOSPER Liposomal Transfection Reagent)
In some systems, increasing the amount of both FuGENE 6 Transfection
Reagent and DNA (more than ten fold higher than the recommended amounts),
can continue to increase the level of protein expression. The very low
cytotoxicity of FuGENE 6 Reagent permits both the FuGENE 6 Reagent and DNA
levels to be tested at these high levels without adversely affecting cell
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