Transfection Basics Support—Getting Started

A wide variety of gene delivery techniques are available to introduce plasmid DNA, siRNA or duplex RNAi, oligonucleotides, and RNA into eukaryotic cells for a variety of research and drug discovery applications. A review of these techniques with pros and cons of each technique is provided here.

Our transfection reagents are shipped under ambient conditions and should be stored at 4°C immediately upon receipt. We guarantee the performance of the product, if stored and handled properly, for one year from date of receipt unless otherwise stated on the tube label or COA. We do not recommend freezing transfection reagents, as this usually decreases transfection performance.

Please see this paper on ambient shipping of Lipofectamine® transfection reagents.

Yes. Cell density will affect transfection performance. Lipofectamine® 3000, Lipofectamine® 2000, and Lipofectamine® LTX/PLUS provide excellent transfection performance at confluencies between 70 and 90%, while some toxicity may be observed at confluencies lower than this. Lipofectamine® RNAiMAX works best at confluencies between 60 and 80%.

Passage number may affect transfection experiments. We recommend consistent splitting and plating of cells. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20–30 passages. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen. Please refer to the Gibco® Cell Culture Basics handbook for proper guidelines for culturing and passaging cells.

No. The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.

The protocol that is supplied with the product will provide you with an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary. 

Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details.

In general, transfection efficiency will show some degree of variability from one transfection to another, no matter how hard one tries to control the parameters of transfection. Keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. To minimize the effect of transfection variability, one can use an internal reference control such as β-galactosidase or luciferase. Co-transfect the expression plasmid with the reference plasmid and assay for the activity of β-gal or luciferase.

Expression in transiently transfected clones is typically higher because transiently transfected cells have a higher copy number of the gene (hundreds per cell). Stably transfected clones usually harbor 1–2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.

Cell line–specific transfection protocols can be found here. If you do not find a cell line–specific protocol or if the protocol does not perform as expected, we recommend testing the conditions described in the protocol supplied with the product to determine the optimal protocol. Successful transfection depends on the cell type, amount of lipid, cell health, passage number, and cell density at the time of transfection. Each of these factors may differ slightly from lab to lab and may require additional optimization of the protocol to achieve the same result. 

Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria. 

It is not necessary to use serum-free media during lipid transfection. However, it is critical to form the lipid:nucleic acid complex in the absence of serum, because proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serum-containing medium.    

Yes, antibiotics can be used in medium during transfection.We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.

Yes, cells can continue to divide up to two times in the presence of lethal doses of G418. The effect of the drug usually becomes apparent by two days.

Yes. The standard transfection protocol may be followed by keeping the total amount of DNA in the mixture constant. That is, if your protocol requires 1 mg plasmid, use 0.5 mg of each of two co-transfected plasmids, or 0.25 mg of each of 4 co-transfected plasmids. When performing co-transfections to introduce a selectable marker on a different plasmid, we recommend using a 3:1 to 10:1 molar excess of the plasmid of interest over the selectable plasmid to ensure that the plasmid of interest is present with the selectable plasmid.

Our cationic lipid transfection reagents can be used to transfect DNA, siRNA, Stealth™ RNAi, mRNA, dicer-generated siRNA pools, or plasmids containing shRNA cassettes. Oligonucleotides, proteins, and RNA can also be transfected. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb. 

You can co-transfect plasmids and siRNA using i) Lipofectamine® 3000 in conjunction with P3000 or ii) Lipofectamine® 2000. 

We recommend using Lipofectamine™ RNAiMAX Reagent for delivery of siRNA into all cell types. It has been specifically developed for siRNA transfection while providing high transfection efficiency with minimal cytotoxicity. As a result, less optimization is necessary. For vector DNA–based RNAi applications, we recommend Lipofectamine™ 3000 Reagent with the P3000™ Enhancer Reagent.

To prepare endotoxin-free DNA, we provide a number of nucleic acid purification kits, the PureLink™ HiPure Purification Kits, in Mini, Midi, Maxi, Mega, and Giga sizes. These kits contain a patented anion-exchange resin to purify plasmid DNA to a level equivalent to 2X CsCl gradients. For more information, please click here. 

1 A260 unit (plasmid DNA in H₂O) = 50 mg/mL. The extinction coefficient will change if the plasmid DNA is diluted in a buffer other than H₂O. This will change the value indicated above.

Sample calculation:

Volume of plasmid DNA sample = 100 mL

Dilution (1/20) = 25 mL of the sample in 475 mL H₂O

A260 of diluted sample = 0.65

Note: For optimal results, make sure OD values are within 0.1 and 1.0.

Concentration of plasmid DNA sample = 0.65 x 50 mg/mL x 20 (dilution factor) = 650 mg/mL

Amount of plasmid DNA in sample = 650 mg/mL x 0.1 mL (sample volume) = 65 mg

An A260/A280 value that is greater than or equal to 1.8 means that the plasmid DNA is pure. A260/A280 readings that are less than 1.8 indicate that the sample may be contaminated with aromatic products (i.e., phenol) or protein. Readouts greater than 2.0 suggest that the sample is contaminated with RNA. 

These are all different cationic-lipid formulations. Lipofectamine® 3000 provides the best transfection performance for both plasmid DNA and RNAi delivery over the broadest range of cell types.  Lipofectamine® LTX was designed for delivery of plasmid DNA with minimal cytotoxicity. Lipofectamine® PLUS is a discontinued transfection reagent, although the PLUS™ Reagent is available and sold separately (Cat. No. 11514-015). Lipofectin® was originally launched in the late 1980s and is considered our very first transfection reagent. We continue to offer these products for customers who prefer the older formulations, but recommend that all new customers try Lipofectamine® 3000 first for optimal performance and lowest toxicity. 

In transient transfection, the transfected DNA does not integrate into the host genome, as a result of which the foreign DNA will be lost at a later stage when the cells undergo mitosis. The expression is short-lived (maximum of 7–10 days) but the level of expression is high, since several copies of the DNA are delivered into the cell. In stable transfection, the transfected DNA integrates into the host genome and therefore remains in the genome of the cells and their daughter cells. The expression is thus sustained as long as the selection pressure is maintained. The expression level is low since only 1–2 copies of the DNA are integrated per cell. Transfection efficiency in a stable transfection is about 1–10% of that in a transient transfection.

The main advantage of lipid-mediated transfection is the higher transfection efficiency that can be achieved with cell types that cannot be transfected using calcium phosphate. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.

Transfection does not work for certain cell types such as non-dividing cells, whereas viral transduction works for dividing as well as non-dividing cells, such as neuronal cells that are hard to transfect.

The dose-response curve is a valuable tool to determine cell toxicity when exposed to various concentrations of antibiotic. The amount of selective antibiotic required to select for resistant cells varies with a number of factors, including cell type and type of antibiotic. We recommend performing a dose-response curve every time a new antibiotic (or a different brand) or a different cell line is used.

Experimental outline of dose-response curve assay:

1. Plate cells in a number of wells such that they are 25–30% confluent. This means that the cells are still dividing and hence will respond well to the antibiotic.
2. Dilute the antibiotic being tested to a broad linear concentration of the recommended range in growth medium.
3. Remove the growth medium from the cells. Apply the antibiotic-containing medium to the respective wells, leaving one set of wells empty. To these wells, add growth medium that does not contain the antibiotic.
4. Culture cells under proper growth conditions (change the medium every 3–4 days to get rid of dead cells and add fresh medium containing antibiotic) and observe the cells daily. At 10–14 days, assess the number of viable cells in each well. (This time period depends upon the antibiotic being tested; antibiotics such as Geneticin®, Hygromycin, and Zeocin™ take about 3 weeks to kill cells, so waiting for 10–14 days would be ideal. However, for Blasticidin, which kills cells in about 2 weeks, waiting for 7–10 days would be sufficient.) To do this, aspirate the medium, wash the cells with phosphate-buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
5. Plot the number of viable cells against the antibiotic concentration. This curve is the dose-response curve or kill curve. The lowest concentration of the antibiotic that kills all the cells in the chosen time period is then used for the stable selection.