SPEED DNA-IN™ Transfection Reagent is a unique formulation of multiple polycations and liposomes that enable highly efficient and effective DNA transfection of eukaryotic cells. DNA-IN™ is the best transfection reagent for over 15 different cell lines, including difficult-to-transfect, suspension, and primary cells.
Size 1 ml
Storage Keep at 4°C. Do not freeze.
Protocol
These conditions are recommended as guidelines only. You may need to do optimization based on your special needs. A good starting point is: DNA(µg): DNA-IN™(µl) ratios should be 1:1 to 1:5. A 6-well or 35 mm dish is adequate for most applications, but larger vessels are sometimes required. In that case, consult table 1 in the attached datasheet.
- Adherent Cells: 18 to 24 hours prior to transfection, seed cells at a density of 1-3 x 10^5 cells per well in 2.0 ml 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 10^5 cells in 0.8 ml 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.
- For each transfection sample, prepare the complexes as follows:
Solution A: Dilute 2.0 µg of DNA into 100 µl of serum-free, antibiotic-free medium.
Solution B: Vortex DNA-IN™ reagent thoroughly prior use, then dilute 10-20 µl of DNA-IN™ in 100µl serum-free, antibiotic-free medium.
- Incubate Solution A and B at room temperature for 5 minutes. Combine the solutions, mix gently to ensure uniform distribution and incubate for 20 minutes at room temperature. (NOTE: Complexes are stable at room temperature for 3-5 hours)
(For suspension cells, go directly to step 5)
- Adherent Cells ONLY: Add 0.8 ml of serum-free, antibiotic-free medium to DNA-IN™-DNA complex. Mix solution gently. Remove growth medium from the cells and add 1.0 ml of DNA-IN™-DNA solution to each well.
- Suspension Cells ONLY: Add 0.2 ml of the DNA-IN™ -DNA solution into each well containing suspension cells in 0.8 ml serum-free, antibiotic-free medium.
- After 5-8 hours, remove transfection solution and add 2.0 ml 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 before assay or making stable cell lines.
|