GIBCO information:
Product: LIPOFECT AMINE Reagent
Cat. No.: 8324SA
Size: 1ml, 2mg/ml
Description
LIPOFECT AMINE Reagent is a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) (Chemical Abstracts Registry name: N-[2-(2,5-bis[(3-aminopropyl)amino]-1-oxpentyl}amino) ethyl]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanaminium trifluoroacetate), and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane filtered water. It is suitable for the transfection of DNA and RNA into culture eukaryotic cells. One mililiter is enough for 60 to 200 transfections on 35-mm tissue culture dishes or 15 to 70 transfections on 60-mm dishes.
Transfection Optimisation
The most important aspect of successful transfection is the careful optimization of transfection conditions for each cell type. GIBCO BRL recommends that the optimal amount of LIPOFECT AMINE Reagent, DNA concentration, cell density and incubation time of LIPOFECT AMINE Reagent-DNA complexes with cells be determined for each cell type. Cell density must be kept consistent to obtain reproducible results. The amount of LIPOFECT AMINE Reagent can be optimized first using a constant amount of DNA. GIBCO BRL recommends starting with 1-2ug DNA for 35-mm culture dishes and a 6 h incubation time. With these two parameters held constant, vary the amount of LIPOFECT AMINE Reagent to determine the optimal concentration (usually 2-25ul). The amount of DNA as well as time of incubation of cells with the LIPOFECT AMINE Reagent-DNA complexes (2-24 hours) can also be optimized. Cell density can be varied to increase efficiency and decrease toxicity. The concentrations of DNA and LIPOFECT AMINE Reagent, cell density and transfection times in the following transient protocols were determined with the plasmids pSV2-CAT and pCMVCAT in BHK-21, HeLa, COS7, CHO, NIH3T3, PC12 and Jurkat cell lines as well as passaged primary human keratinocytes and fibroblasts. Stable tranformations were performed in NIH3T3 cells. These conditions are recommended as guidelines only.
Procedure for transient or stable transfection of adherent cells
1. In a six-well or 35mm tissue culture plate, seed ~ 1-3 x 105 cells per well in 2ml of the appropriate growth medium supplemented with serum.
2. Incubate the cells at 37¡C in a CO2 incubator until the cells are 50-70% confluent. This will usually take 18-24h, but the time will vary among cell types. (NOTE: For the above cells evaluated, 50-70% confluence at the time of transfection was optimal, however for other cell lines, optimal cell density may vary. Since transfection efficiency is sensitive to culture confluence, it is important to maintain a standard seeding protocol from experiment to experiement. Note **70-80% is optimal for COS cells).
3. Prepare the following solutions in 12 X 75mm sterile tubes:
Solution A: For each transfection, dilute 1-2ug of DNA into 100ul serum-free medium. OPTI-MEM, I Reduced Serum Medium (GIBCO BRL Cat. No. 320-1985) gives optimal results.
Solution B: For each transfection, dilute 2-25ul of LIPOFECT AMINE Reagent into 100ul serum-free medium. (NOTE: When transfecting with higher amounts of DNA and LIPOFECT AMINE Reagent use larger volumes of serum-free medium to dilute the DNA and LIPOFECT AMINE Reagent. When using larger tissue culture plates, increase the amounts of all reagents in proportion to the surface area.
For COS7 cells 16ul per 3 cm dish of near confluent cells (1.6 X 105 cells grown o/n is optimal). ). See specific entry for Cos cells.
4. Combine the two solutions, mix gently, and incubate at room temperature for 15-45 min. The solution may appear cloudy, however this will not impede the transfection.
5. Wash the cells once with 2ml of serum-free medium.
6. For each transfection, add 0.8ml of serum-free medium to each tube containing the lipid-DNA complexes. Medium containing 5% serum may be added to the DNA-lipid complexes at this step (see Note No.3). Do not add antibacterial agents to media during transfection. Mix gently and overlay the diluted complex solution onto the washed cells.
7. Incubate the cells for 2-24h at 37¡C in a CO2 incubator. GIBCO BRL recommends starting with 6 h.
8. Add 1ml of growth medium containing twice the normal concentration of serum without removing the transfection mixture. If serum was included previously, add 1ml of normal growth medium at this time. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium.
9. Replace medium at 18-24h following start of transfection.
10. Assay cell extracts for gene activity 24-72h after the start of transfection, depending on cell type and promoter activity.
11. This same procedure can be used to transfect DNA for stable expression. Instead of harvesting cultures 72h post tranfection, passage 1:10 into the selective medium for the reporter gene transfected. For instance, medium should contain GENETICIN, GIBCO BRL (Cat. No. 11811-031) for pRSV neo transfections.