Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
INTRODUCTION OF DNA INTO CELLS
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Procedure for Transient or Stable Transfection of Adherent Cells using Lipofectamine |
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| Contributor: |
Members of the Laboratory of David Bowtell: C. Andrews, F. Christensen, N. Della, D. Donald, C. Fernandez, A. Holloway, Y. Hu, M. Kafali, R. Parkinson, H. Robertson, D. Wang. and V. Hammond at the University of Melbourne. URL: Lab Web Site | ||||||||||||||||
| Overview |
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| The most important aspect of successful transfection is the careful optimization of transfection conditions for each cell type. Empirically determine the optimal amount of LIPOFECT AMINE Reagent, DNA concentration, cell density and incubation time of LIPOFECT AMINE Reagent-DNA complexes with cells for each cell type. | |||||||||||||||||
| Procedure |
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1. In a six-well or 35 mm tissue culture plate, seed approximately 1 to 3 X 105 cells per well in 2 ml of the appropriate growth medium supplemented with serum. 2. Incubate the cells at 37°C in an incubator until the cells are 50 to 70% confluent. This will usually take 18 to 24 hours, but the time will vary among cell types (see Hint #2). 3. Prepare the following two solutions in 12 x 75 mm sterile tubes: a. For each transfection, dilute 1 to 2 μg of DNA into 100 μl of serum-free medium (see Hint #3). b. For each transfection, dilute 2 to 25 μl of LIPOFECT AMINE Reagent into 100 μl of serum-free medium (see Hint #4). 4. Combine the two solutions, mix gently, and incubate at room temperature for 15 to 45 min. The solution may appear cloudy. 5. Wash the cells once with 2 ml of serum-free medium. 6. For each transfection, add 0.8 ml of serum-free medium to each tube containing the lipid:DNA complexes (see Hint #5). Do not add antibacterial agents to media during transfection. Mix gently and overlay the diluted lipid:DNA solution onto the washed cells. 7. Incubate the cells for 2 to 24 hours at 37°C in an incubator. 8. Add 1 ml of growth medium containing twice the normal concentration of serum without removing the transfection mixture. If serum was included in step 6, add 1 ml 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 to 24 hours following start of transfection. 10. Depending on cell type and promoter activity, assay cell extracts for gene activity 24 to 72 hours after the start of transfection. (See Hint #6) |
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| Solutions |
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| BioReagents and Chemicals |
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Lipofect Amine Reagent |
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| Protocol Hints |
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1. 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 cultured eukaryotic cells. One milliliter is enough for 60 to 200 transfections on 35-mm tissue culture dishes or 15 to 70 transfections on 60-mm dishes. 2. For HeLa, BHK-21, CHO, NIH3T3, PC12, and Jurkat cell lines, 50 to 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 experiment. For example, 70 to 80% confluency is optimal for COS cells. 3. OPTI-MEM, Reduced Serum Medium gives optimal results. 4. This amount of LIPOFECT AMINE reagent to use is given as a suggestion. For example, for COS7 cells, 16 μl of LIPOFECT AMINE reagent per 3 cm dish of near confluent cells is optimal. The amount of reagent you use should be determined for your specific cell line. Use a constant amount of DNA and 6 hour incubation to first optimize the amount of LIPOFECT AMINE reagent to use. The amount of DNA and incubation time can then be optimized. Cell density can be varied to increase efficiency and decrease cell toxicity. However, once determined, cell density must be kept constant between experiments to achieve consistent results. 5. Media containing 5% serum may be added to the DNA:lipid solution at this step. 6. This same procedure can be used to transfect DNA for stable expression. Instead of harvesting cultures 72 hours post-transfection, passage 1:10 into selective medium for the reporter gene transfected. |
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