Ordering Information:
|
FT101-01
|
0.75 ml
|
|
FT101-02
|
2×0.75 ml
|
Storage: at 2-8oC for one year (avoid freezing)
Description
TransLipid® Transfection Reagent is a proprietary formulated cationic lipid that offers superior transfection efficiency across a broad range of mammalian cell lines.
• Transfect DNA and/or RNA.
• Adherent or suspension cells.
• Can be used in the presence of serum.
Applications
Transfection of adherent cells and suspension cells (mammalian cell lines)
For most cells, it is suggested that ratio of DNA to translipid is 1:2-1:3. To obtain better transfection efficiency, we suggest to use high density cell (70-90% confluency).
Plasmid DNA Transfection
Use 24-well format as example. To transfect cells in other formats.
1. Adherent cells: One day before transfection, plate 0.5-2×105cells in 500 μl of growing medium without antibiotics so that cells will be 70-90% confluent at the time of transfection.
Suspension cells: Just prior to preparing complexes, plate 4-8×105 cell in 500 μl of growing medium without antibiotics.
2. For each transfection, prepare complexes as follows
a. Dilute 0.8 μg DNA in 50 μl of Opti-MEM I Reduced Serum Medium without serum in one tube. Mix gently.
b. Mix TransLipid® gently before use, then dilute 2 μl TransLipid® in 50 μl of Opti-MEM I Medium in another tube. Mix gently. Incubate for 5 minutes at room temperature.
c. After the 5 minutes incubation, combine the diluted DNA with diluted TransLipid® . Mix gently and incubate for 20 minutes at room temperature. DNA- TransLipid® complex is stable at room temperature for 6 hours.
3. Add the DNA- TransLipid® complexes to each well containing cells and medium. Mix gently by shaking the plate back and forth.
4. Incubate cells at 37oC in a CO2 incubator for 18-48 hours prior to testing for gene expression. Medium may be changed after 4-6 hours.
5. For stable cell lines: Passage cells at 1:10 into fresh growth medium 24 hours after transfection. Add selective medium in the following day for selection.
Optimizing Plasmid DNA Transfection
In order to achieve the maximum transfection efficiency and minimize the effect of cytotoxicity, suggest to test DNA (μg) to Lipid (μl) in the range of 1:0.5 to 1:5 of DNA (μg) to TransLipid®(μl).
siRNA Transfection
1. One day before transfection, plate cells in 500 μl of growing medium without antibiotics so that cells will be 30-50% confluent at the time of transfection.
2. For each transfection, prepare complexes as follows:
a. Dilute 20 pmol siDNA oligomer in 50 μl of Opti-MEM I Reduced Serum Medium without serum. Mix gently.
b. Mix TransLipid® gently before use, then dilute 1 μl TransLipid® in 50 μl of Opti-MEM I Medium. Mix gently and incubate for 5 minutes at room temperature.
c. After the 5 minutes incubation, combine the diluted DNA with diluted TransLipidTM . Mix gently and incubate for 20 minutes at room temperature.
3. Add the siRNA oligomer- TransLipid®complexes to each well containing cells and medium. Mix gently by shaking the plate back and forth.
4. Incubate cells at 37oC in a CO2 incubator for 24-96 hours until you are ready to assay for gene expression. Medium may be changed after 4-6 hours.
In order to achieve the maximum transfection efficiency and minimize the effect of non-specificity, the concentration of siRNA and TransLipid®could be optimized. For transfection in a 24-well plate, it is recommended to use a range of 10 to 50 pmol of siRNA and 0.5 to 1.5 μl ofTransLipid® . Based on the property of different target genes, increasing the cell density for transfection can be taken into consideration of condition optimization.
