DNAfectin™ Transfection Reagent, 1 ml - Must be stored at -20° C
- Note: Additional Shipping Charges of $25 Apply
Description DNAfectin
TM comprises of a unique formulation of polycations and liposomes, which will
guarantee high transfection efficiency and low cytotoxicity for any cell type including
primary cells. Over 50% transfection efficiency can be achieved using our DNAfectin
TM formulation for any cell line.
Transfection Protocol Use the following conditions as guidelines to transfect mammalian cells in a 6-well
or 35mm dish format. For other culture vessels, please refer to Table 1.
1. Adherent Cells: 18 to 24 hours prior to transfection, seed cells at a density of
1-3 x 105 cells per well in 2.0ml of appropriate growth medium (with serum and
antibiotics if cells are cultured in the presence of them). Incubate the cells
at 37˚C in a CO2 incubator until cells are 70% to 90% confluent at the time of
transfection.
Suspension Cells: Just prior to preparing complexes, plate 3-5 x 105 cells in 0.8ml of
serum free medium without antibiotics.
Since transfection efficiency is sensitive to culture confluence, it is important to
maintain a standard seeding protocol from experiment to experiment.
2. For each transfection sample, prepare the complexes as follows:
Solution A: Dilute 2.0μg of DNA into 100μl of serum-free, antibiotic-free medium.
Solution B: Vortex DNAfectin
TM reagent thoroughly prior use, then dilute 10-20 μl
of DNAfectin
TM reagent in 100μl serum-free, antibiotic-free medium.
Incubate Solution A and B at room temperature for 5 minutes.
3. Combine the solutions, mix gently to ensure uniform distribution and incubate for
20 minutes at room temperature. For suspension cells, go directly to step 5.
NOTE: Complexes are stable at room temperature for 3-5 hours.
4. Adherent Cells ONLY: Add 0.8ml of serum-free, antibiotic-free medium to
DNAfectin
TM -DNA complex. Mix solution gently.
5. Adherent Cells: Remove growth medium from the cells and add 1.0ml of
DNAfectin
TM -DNA solution to the each well containing cells.
Suspension Cells: Add 0.2ml of the DNAfectin
TM -DNA solution into each well
containing suspension cells in 0.8ml serum-free, antibiotic-free medium.
6. After 5-8 hours, remove transfection solution and add 2.0ml of the appropriate
growth medium (with serum and antibiotics) or add 0.1ml of FBS directly into
each vessel.
Incubate the cells at 37˚C in a CO2 incubator for a total of 18-24 hours.
7. To make stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh
growth medium 24 hours post transfection. Selection medium can be added the
following day if desired.
Optimizing Transfection for Specific Cell Lines
To achieve the maximum transfection efficiency and low cytotoxicity, optimize
transfection conditions by varying cell density along with DNA and DNAfectinTM
concentrations. Optimal results have been observed when cells were 80-90%
confluent and DNA(μg): DNAfectinTM (μl) ratios were 1:1 to 1:5.
| Culture Vessel | Surface area per well (cm2) | Volume of plating medium | DNA(µg) in medium volume (µl) | DNAfectinTM in medium volume | Transfection medium vol. |
| 24-well | 2 | 500 µl | 0.2-0.4 µg in 25 µl | 2-4 µg in 25 µl | 0.4 ml |
| 12-well | 4 | 1 ml | 0.5-0.8 µg in 100 µl | 5-8 µg in 100 µl | 0.6 ml |
| 6-well | 10 | 2 ml | 1.0-2.0 µg in 100 µl | 10-20 µg in 100 µl | 0.8 ml |
| 35 mm | 10 | 2 ml | 1.0-2.0 µg in 100 µl | 10-20 µg in 100 µl | 0.8 ml |
| 60 mm | 20 | 5 ml | 3.0-6.0 µg in 500 µl | 30-75 µg in 500 µl | 2.4 ml |
| 10-cm | 60 | 10 ml | 8.0-16.0 µg in 1.5 ml | 90-200 µg in 800 µl | 6.4 ml |
Equipment 