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:6/27/2016)... 27, 2016   Ginkgo Bioworks , a leading ... was today awarded as one of the World ... world,s most innovative companies. Ginkgo Bioworks is engineering ... real world in the nutrition, health and consumer ... with customers including Fortune 500 companies to design ...
(Date:6/24/2016)... ... ... Researchers at the Universita Politecnica delle Marche in Ancona combed medical journal ... Their findings are the subject of a new article on the Surviving Mesothelioma website. ... the blood, lung fluid or tissue of mesothelioma patients that can help point doctors ...
(Date:6/23/2016)... 23, 2016 /PRNewswire/ - FACIT has announced the ... biotechnology company, Propellon Therapeutics Inc. ("Propellon" or ... of a portfolio of first-in-class WDR5 inhibitors for ... as WDR5 represent an exciting class of therapies, ... medicine for cancer patients. Substantial advances have been ...
(Date:6/23/2016)... SANTA MONICA, Calif. , June 23, 2016  The Prostate Cancer ... to pioneer increasingly precise treatments and faster cures for prostate cancer. Members of ... 77 institutions across 15 countries. Read More About the ... ... ...
Breaking Biology Technology:
(Date:6/27/2016)... Research and Markets has announced the addition ... to their offering. The report ... to grow at a CAGR of 12.28% during the period 2016-2020. ... in-depth market analysis with inputs from industry experts. The report covers ... The report also includes a discussion of the key vendors operating ...
(Date:6/22/2016)... -- The American College of Medical Genetics and Genomics was once ... one of the fastest-growing trade shows during the Fastest 50 ... Las Vegas . Winners ... each of the following categories: net square feet of paid ... The 2015 ACMG Annual Meeting was ranked 23 out of ...
(Date:6/22/2016)... 22, 2016   Acuant , the ... solutions, has partnered with RightCrowd ® ... Visitor Management, Self-Service Kiosks and Continuous Workforce ... add functional enhancements to existing physical access ... venues with an automated ID verification and ...
Breaking Biology News(10 mins):