Optimization Tips

Refer to the following table if you observe low transfection efficiency.

Possible Cause Recommendation 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 container. 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 additives

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 1 2 3 4 5 6 Add serum-free medium (l) 97 97 97 94 94 94 Add FuGENE 6 Transfection Reagent (l) 3 3 3 6 6 6

Add DNA (g)

0.5 1 2 1 2 3

1. Gently tap the tubes. Mix thoroughly, but do not vortex.
2. Incubate at room temperature for 15-45 minutes.
3. 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.
4. If you increase the DNA concentration (e.g., in a co-transfection experiment), proportionally increase the amount of FuGENE 6 Transfection Reagent.
5. 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.
6. If no transfection is observed, repeat the experiments with DOTAP or DOSPER Liposomal Transfection Reagent)

Amount of Transfection complex was too low 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 viability.

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