Calcium Phosphate Transfection Kit - FAQs

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9 product FAQs found

I have tried several different transfection reagents and have failed to transfect my gene into my cell line of interest. Do you have any suggestions?

We recommend that you try electroporation as a method of delivering your plasmid of interest. We offer the Neon Transfection System for highly efficient transfection of primary cells, stem cells, and difficult-to-transfect cells. You may also consider using a viral-based system (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/viral-delivery-mammalian-expression.html) to deliver your gene into your mammalian cell line of interest.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

How do I perform a dose-response curve or kill curve?

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of viral transduction over transfection?

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of lipid-mediated transfection over calcium phosphate-mediated 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. Calcium phosphate is prone to variability due to its sensitivity to slight changes in pH, temperature, and buffer salt concentrations. Calcium phosphate may also be cytotoxic to many cell types, especially primary cells. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main difference between transient and stable transfection?

During transient transfection the exogenous DNA does not integrate into the host genome, as a result some DNA is lost with every subsequent cell division. The expression is short-lived (maximum of 7-10 days) but the level of expression is high, since up to hundreds of copies of the DNA may be delivered into the cell. In stable transfection, under antibiotic selection pressure, the DNA integrates into the host cell genome and is passed onto their daughter cells during cell division. 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 may be integrated per cell. Transfection efficiency in a stable transfection is about 1-10% of that in a transient transfection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do you estimate the amount of DNA using A260 readings? What does A260/A280 mean?

1 A260 unit (double stranded DNA in H2O) = 50 mg/mL. The extinction coefficient will change if 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 between 1.8 and 2.0 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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use the same amount of any transfection reagent for different cell lines?

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. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For additional troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport)

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What recommendations do you have for selecting a transfection reagent?

Choose the best reagent by cell type and application by using the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What are the different methods available for transfection?

There are many transfection methods available to deliver plasmids, DNA fragments, oligos, siRNAs, mRNA, or proteins for a wide range of research and drug discovery applications. A review of the pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.