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How is the DNA:lipid reagent ratio calculated for Promega's TransFast™, Tfx™
and Transfectam® Reagents?
The amount of positive charge contributed by the cationic lipid component of the
transfection reagent should equal or exceed the amount of negative charge contributed by
the phosphates on the DNA backbone, resulting in a net neutral or positive charge on the
multilamellar vesicles associating with the DNA. This ratio is generally in the range of
1:1 to 2:1 for TransFast™ Reagent(*) (Cat.# E2431), 2:1 to 4:1
for Tfx™ Reagents(*)
(Tfx™-50 Reagent: Cat.#
E1811; Tfx™-20 Reagent: Cat.# E2391 ;
Tfx™-10 Reagent: Cat.#
E2381) and 4:1 and greater for Transfectam®
Reagent(*) (Cat.# E1231, E1232).
However, this ratio should be optimized for each reagent/cell line combination. The same
cell line may even have different optimum ratios when grown under different conditions.
Promega's three Tfx™ Reagents (see Note below) contain the
same concentration of the synthetic cationic lipid N, N, N´, N´- tetramethyl-N,
N´,-bis(2-hydroxyethyl)-2,3,-dioleoyloxy-1,4-butanediammonium iodide and different molar
ratios of the fusogenic lipid, DOPE. TransFast™ Transfection Reagent also contains
the fusogenic lipid DOPE, but contains the synthetic cationic lipid N,N [bis
(2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy) propyl] ammonium iodide at a defined
molar ratio. Transfectam® Reagent only consists of a synthetic cationic
polyamine called dioctadecylamidoglycyl spermine (DOGS). It does not contain DOPE. When
resuspended as described in the respective technical bulletins, TransFast™ Reagent
and Tfx™ Reagent contain 1nmol/µl of the synthetic cationic lipid, while the
Transfectam® Reagent has 2nmol/µl. All of these lipids differ in the number
of positive charges per molecule, and thus the volume of reagent needed to make a 1:1
molar charge ratio with 1µg of DNA also differs accordingly.
Note: We also offer the Tfx™ Reagents Trio (Cat.#
E2400), a starter set of all three Tfx™ Reagents for determining which reagent is
best for your cell line and culture conditions.
Table 1 lists the the volume of transfection reagent needed to form a 1:1 charge ratio
of reagent:DNA for each reagent type.
Table 1. Summary of Promega's
Different Synthetic Cationic Lipid Transfection Reagents and the Amounts Needed for a 1:1
Charge Ratio with 1µg of DNA. |
|
TransFast™
Reagent |
Tfx™ Reagents |
Transfectam® Reagent |
Positive charges per molecule cationic lipid |
1 |
2 |
4 |
Moles cationic lipid per microliter |
1nmol |
1nmol |
1.97nmol |
Moles charge per microliter |
1nmol |
2nmol |
8nmol |
Volume reagent needed for 1:1 with a microgram
of DNA |
3µl |
1.5µl |
0.375µl |
Recommended starting ratios (reagent:DNA) |
1:1 to 2:1 |
2:1 to 4:1 |
4:1 to 13.3:1 charge ratio (1.5µl to 5µl/µg DNA) |
Note that each microgram of DNA has 3nmol of negative charges.
Tfx and TransFast are trademarks of Promega Corporation.
Transfectamis a registered trademark of BioSepra, S.A., the holder of a license from
CNRS-ULP Strasbourg under U.S. Pat. No. 5,171,678 to sell the Transfectam®
product for research purposes only. The Transfectam® product was developed by
J.P. Behr and J.P. Loeffler and is covered by the aforementioned patent.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
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