Navigation Links
Uses and Applications of FuGENE 6 Transfection Reagent

Troubleshooting Hints if You Observe Cytotoxicity (greater than 5 - 10 % Cell Death)

FuGENE 6 Transfection Reagent has proven to be virtually nontoxic to most cell types. If you observe more than 5 - 10 % cell death in transient transfection experiments, or if selection of permanent transfectants fails, refer to the table below for possible causes.

Possible Cause Recommendation Selection antibiotic added too soon

Repeat transfection and wait an additional 24 to 48 h before adding the selection antibiotic to allow for sufficient expression of the selection marker. Selection antibiotic concentration too high

Repeat transfection using a lower concentration of selection antibiotic. Transfected protein is cytotoxic or is produced at excessively high levels Reduced viability or slow growth rates may be the result of high levels of protein expression, as the cells metabolic resources are directed toward the production of the heterologous protein. The expressed protein may also be toxic to the cell at the level expressed. To analyze cytotoxicity, prepare experimental controls as described below.

Prepare extra wells containing:

  1. Cells that are not transfected.
  2. Cells transfected with DNA alone (i.e., without FuGENE 6 Transfection Reagent)
  3. Cells treated with FuGENE 6 Transfection Reagent alone (no DNA added).

Compare cells transfected with the experimental construct to wells that contain these experimental controls. Consider repeating th e experiment with a secreted reporter gene assay such as SEAP, hGH, or a standard -gal control vector. Cells secreting SEAP should show little to no evidence of cytotoxicity.

The culture may be contaminated with mycoplasma

Use the Mycoplasma Detection Kit (Cat. No. 1 296 744) or Mycoplasma PCR ELISA (Cat. No. 1 663 925) to determine if the culture is contaminated. Treat the cells with BM Cyclin (Cat. No. 799 050) to eliminate the mycoplasma. Alternatively, start the transfections over with a fresh uncontaminated culture.

Cells may not be healthy (e.g., malfunctioning incubator, media problems)

Assess the physiological state of the cells and incubation conditions (e.g., CO2 and temperature levels). Perform the same controls as suggested above for cytotoxicity to eliminate the influence of transfection reagent or nucleic acid. Plasmid preparation
contaminated with large amounts of endotoxin


Endotoxin is reported to be cytotoxic to some very sensitive cell lines (e.g., Huh-7) and primary cultures. By using FuGENE 6 Transfection Reagent for many common cell types, it may be possible to use DNA containing higher endotoxin levels. If above tests prove negative, FuGENE 6 Transfection Reagent may be cytotoxic to your specific cell type If you are using a very sensitive cell line, measures can be taken to minimize cytotoxicity:
  • Perform the transfection in the presence of FBS
  • Reduce the time of exposure to the transfection reagent:DNA complex to 23 h, then replace the medium
  • Perform the transfection at a highe r cell density
  • Use different ratios of FuGENE 6 Transfection Reagent to DNA


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. A Versatile Power Supply for All Electrophoresis Applications
2. Highest Possible Transformation Efficiencies for High-Throughput Applications
3. Unique DNA Polymerase Formulation Excels in a Broad Range of PCR Applications
4. High-Throughput System Generates Ultra-Pure PCR Products Suitable for All Applications
5. Optimizing Büchi® Rotary Evaporator Applications
6. Optimizing Büchi® Rotary Evaporator Applications
7. Optimizing Bchi Rotary Evaporator Applications
8. Uses and Applications of X-tremeGENE Q2 Transfection Reagent
9. Uses and Applications of FuGENE 6 Transfection Reagent
10. Uses and Applications of FuGENE 6 Transfection Reagent
11. Uses and Applications of FuGENE 6 Transfection Reagent
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:4/27/2017)... ... April 27, 2017 , ... This month Quorum ... solution that simplifies research studies, accelerates study startup, and improves participant engagement. Developed ... is the first and only IRB-integrated eConsent solution . , “Our mission ...
(Date:4/27/2017)... (PRWEB) , ... April 27, 2017 , ... ... a practicing internist, who will review how testing for 1,25-Dihydroxyvitamin D can enhance ... ordering of 25-OH-vitamin D and 1,25 dihydroxyvitamin D. , Dr. Gregory Plotnikoff, senior ...
(Date:4/27/2017)... ... April 27, 2017 , ... ... name change to Fluence Analytics. , Fluence Analytics provides proprietary hardware ... biopharmaceutical manufacturing processes and R&D applications. The company’s patented technologies improve production ...
(Date:4/27/2017)... (PRWEB) , ... April 27, 2017 , ... ... the 2017 Borlaug CAST Communication Award goes to Jayson Lusk, a consummate communicator ... forms of media to advocate for science, as he explains how innovation and ...
Breaking Biology Technology:
(Date:4/11/2017)... N.Y. , April 11, 2017 ... fingerprints, but researchers at the New York University ... College of Engineering have found that partial similarities ... security systems used in mobile phones and other ... thought. The vulnerability lies in the ...
(Date:4/5/2017)... KEY FINDINGS The global market ... CAGR of 25.76% during the forecast period of 2017-2025. ... for the growth of the stem cell market. ... MARKET INSIGHTS The global stem cell market is segmented ... The stem cell market of the product is segmented ...
(Date:3/30/2017)... Trends, opportunities and forecast in this market ... (fingerprint, AFIS, iris recognition, facial recognition, hand geometry, vein ... use industry (government and law enforcement, commercial and retail, ... others), and by region ( North America ... Pacific , and the Rest of the World) ...
Breaking Biology News(10 mins):