50% OFF Transfectamine™ 5000

Half Price Transfection Reagent

Coupon code T50 must be provided at the time of sale for promotion to apply. Offer only applies to purchases of transfection reagent. Offer expires on 30th April 2020.

Half Price Transfection Reagent

Figure 1. Transfection efficiency comparison in HeLa cells using Transfectamine™ 5000, Lipofectamine 2000 and Lipofectamine 3000 reagents. Each reagent was used to transfect HeLa cells in a 96-well format, and GFP expression was analyzed 24 hours post-transfection. Transfectamine™ 5000 transfection reagent provided higher GFP transfection efficiency compared to Lipofectamine 2000 and Lipofectamine 3000 reagents.

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Cell Lipofection Mechanism

Cell lipofection, sometimes referred to as ‘lipid transfection’ is the method of nucleic acid or protein delivery into cells using a lipid complex to cross the cell membrane, via either endocytosis or fusion. A great deal of optimization is necessary, as the procedure efficiency varies widely based on cell line, biological payload cytotoxicity, and other factors.

Lipofection Procedure Recommendations

Half Price Transfection Reagent

Cell Line Optimization

• Determine cell line transfection resistance
• Older reagent formulations have longer protocols, but more documentation of cell line compatibility
• Suspended cells often require higher densities than adherent cultures
• Scientific support and assistance from reagent manufacturer

Half Price Transfection Reagent

DNA Purity and Quality

• Compare DNA/Reagent concentrations for best performance
• Test different incubation periods of DNA and reagent before cell mixing
• Endotoxin levels should be kept as low as possible
• A260/A280 of 1.7-1.9 are ideal purity levels for most procedures

Half Price Transfection Reagent

Cell Confluency and Health

• An initial cell viability assay to ascertain cell health
• 70-90% confluency recommended for most cell lines
• Minimize required reagent to keep toxicity low
• Prepare cell culture 24 hours prior to procedure for optimal replication state

Example Protocol

Transfectamine™ 5000 Reagent used

1. Prepare cells for transfection

• Culture cells to ~90% confluency and use fresh, clean growth medium

2. Prepare Transfectamine™ 5000-DNA mixture

• First mix 2.5 ug of DNA with 200 uL of serum-free medium.
• Add 7.5 uL of thawed Transfectamine™ 5000 to DNA/medium mixture.
• Mix well and incubate at room temperature for 20 minutes.

Half Price Transfection Reagent

3. Add Transfectamine™ 5000-DNA mixture to cell culture

4. Culture overnight

5. Analyze transfection efficiency with appropriate method

• Maximum expression levels can be discerned 72-96 hours post-transfection
• If using Flow Cytometry, Laser-Scanning Molecular Imaging, Microscopy, or Western Blot Analysis, a fluorescent label is necessary or strongly recommended

Common Issues and Solutions

Problem: Dead or Ailing Cells

• Run an additional control of DNA without reagent: expressed protein may be toxic

• Check for contamination and treat as necessary, or run procedure with new culture

• Selection antibiotic may be too strong or added too soon after procedure. Test again with lower concentrations and longer interval

Problem: Transfection Efficiency Low

• Reagent may not have been stored correctly. Check to ensure that accidental refreezing did not occur

• DNA quality or amount may have been insufficient. Determine appropriate lipid-to-DNA ratio empirically

• Cell medium may be contaminated with serum or antibiotics. Be sure that medium contains no growth enhancers or other additives

Determining Transfection Efficiency With Flow Cytometry

• Use an untransfected control and subtract the baseline intensity data from your experimental results

• Signal intensity graphs are a simple visual aid to compare the response of the cell line to different transfection reagents

• Many instruments have software with FACS analysis programs for quick results

Signal intensity graphs made using flow cytometry analysis of control CHO-K1 cells vs CHO-K1 cells transfected using TransfectamineTM 5000 and other lipofection reagents

Half Price Transfection Reagent

Resources and Support

Half Price Transfection Reagent

• Scientific support and protocol assistance

• Background research and in-house testing to ensure product quality

Assaywise Letters published biannually and Application Notes publicly available for in-depth implementation


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