Navigation Links
Uses and Applications of FuGENE 6 Transfection Reagent

Protocol Instructions for Users Who are New to Transfection

Below, a standard protocol for the DNA transfection of cultured cells using FuGENE 6 is presented that has been specially designed for scientists who are new to transfection.

1. Prepare cells the day before transfection
  • Ensure the cells are growing well and are in log phase. Occasionally cells slow down when they reach confluence, and it can take a day or two to recover.
  • For monolayers, plate the cells so they will only be 50-80% confluent the following day.

  • Plate the cells in medium that does not contain antibiotics. Transfection is lower in some cell lines when they are grown in antibiotics. Possible explanations are that the transfection reagent may bring the antibiotic into the cell and decrease transfection, or the complex may interact with the antibiotic, resulting in less transfection.
  • For most adherent cell lines, plate 100,000-300,000 cells in 2 ml medium (50,000 to 150,000 cells/ml) in one well of a 6-well plate. Plan to test at least 3 ratios of reagent to DNA.. See the table below for a suggested layout of a 6-well plate.
(A) Cell Control

    No reagent

    No DNA
(B) Reagent Control

    6 l reagent

    No DNA
(C) DNA-Control

    No reagent

    1 g DNA
(D) 3:1 ratio

    3 l reagent

    1 g DNA
(E) 3:2 ratio

    3 l reagent

    2 g DNA
(F) 6:1 ratio

    6 l reagent

    1 g DNA

2. Prepare the complex example given for 6-well plate

Prepare the DNA



  • Select a plasmid that you know transfects your cell line, and make sure you are able to measure the expression of reporter gene or transfected protein. Plasmids with reporter genes are available from Roche Applied Science.
  • Handle the DNA as aseptically as possible to avoid contaminating your cell cultures during the transfection. Use sterile pipettes, tips, vials, etc.
  • Accurate DNA concentrations are important. Determine the concentration of the DNA with a spectrophotometer. Transfection-grade DNA should have a 260/280 ratio of 1.8.
  • Check the stock DNA concentration, and determine how much you will need for 1 and 2 g. If the stock is 2 g/l, then decide if you can accurately pipet 0.5 l. It might be better to dilute the DNA using procedures that give accurate dilutions. If you need to dilute the DNA, use sterile DNase-free water or sterile DNase-free TE buffer as the diluent.
  • Calculate accordingly for your DNA concentrations (i.e., concentrations of 0.5 g/l work well; for 1 g, measure 2 l, and for 2 g, measure 4 l).
Prepare the FuGENE 6 Transfection Reagent

  • Remove the FuGENE 6 Reagent from the refrigerator or freezer and allow it to warm to room temperature. Meanwhile, look at the cells microscopically. Make sure they look healthy and are 50-80% confluent. Return the cells to the incubator.
  • Warm serum-free DMEM (no additives) to room temperature. Aliquot 94-100 l into each of 6 tubes as indicated below. Use sterile Eppendorf tubes or small sterile polystyrene tubes labeled:
    • A (100 l)
    • B (94 l)
    • C (100 l)
    • D (97 l)
    • E (97 l)
    • F (94 l), corresponding to the plate layout above.
  • Mix the room-temperature FuGENE 6 Reagent by tapping or briefly vortexing (1 second).
    Carefully add the FuGENE 6 Reagent to the serum-free medium as follows.
    • 6 l to Tube B
    • 3 l to Tube D (3:1 ratio)
    • 3 l to Tube E (3:2 ratio)
    • 6 l to Tube F (6:1 ratio)
  • Ensure the entire amount of reagent is delivered directly from the pipette tip into the serum-free medium. Avoid contact of the FuGENE 6 Transfection Reagent with the walls of the tube containing the serum-free medium. Avoid a FuGENE 6 Reagent layer on top of the serum-free medium. Once the FuGENE 6 Reagent is added, immediately tap, flick, or vortex for 1 second to ensure adequate mixing of the components.
  • Incubate at room temperature for 5 minutes.

Make the complex

Add the DNA to each tube (see table below). The amount added will depend on the starting concentration of DNA.

Tube DNA at 0.5 g/l DNA at 1 g/l DNA at 2 g/l
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Uses and Applications FuGENE Transfection Reagent

(Date:10/1/2014)... September 30, 2014 New applications of ... that have evolved in nature over millennia are featured ... materials for applications in biophotonics in the October ... The journal is published by SPIE, the international society ... Several of the peer-reviewed articles are accessible via open ...
(Date:10/1/2014)... 01, 2014 The U.S. Department of ... Illinois University Edwardsville as part of the Trade Adjustment ... funding is co-administered by the Department of Labor and ... of Labor Thomas E. Perez and Secretary of Education ... job-driven training grants to nearly 270 colleges across the ...
(Date:9/30/2014)... a well-known optical phenomenon called thin-film interference, a ... the ability to "paint" ultra-thin coatings onto a ... making future, flexible electronic devices, creating advanced solar ... ships and spacecraft with extremely lightweight decorative logos. ... it becomes iridescent, revealing a myriad of colors ...
(Date:9/30/2014)... Sept. 30, 2014 Report Details ... highest potential revenues Do you want to find ... you revenue predictions for those biological drugs from ... you find financial data, R&D trends, opportunities and ... sales forecasts to 2024 at overall world market, ...
Breaking Biology Technology:Biomimetic and bioinspired materials enable new healthcare options, reports Journal of Biomedical Optics 2Biomimetic and bioinspired materials enable new healthcare options, reports Journal of Biomedical Optics 3U.S. Dept. of Labor Awards SIUE Nearly $10-Million Grant 2U.S. Dept. of Labor Awards SIUE Nearly $10-Million Grant 3U.S. Dept. of Labor Awards SIUE Nearly $10-Million Grant 4Taking thin films to the extreme 2Biosimilars and Follow-On Biologics: World Industry and Market Prospects 2014-2024 2Biosimilars and Follow-On Biologics: World Industry and Market Prospects 2014-2024 3Biosimilars and Follow-On Biologics: World Industry and Market Prospects 2014-2024 4Biosimilars and Follow-On Biologics: World Industry and Market Prospects 2014-2024 5
... Dec. 13 BioMed Realty Trust, Inc.,(NYSE: BMR ... to its,board of directors, increasing the board to eight ... real estate industry, with a breadth of,ability and experience ... real estate projects. We believe that,Rick,s contributions to our ...
... new validation studies,were published during 2007 on the ... number of studies published to 65., "This ... interest from US and,European institutes" said Gary Zamler, ... academic medical institutions and teaching hospitals,have completed studies ...
... retired $12.83 million in existing current 2008 convertible notes, and extended the ... ... convertible notes, SEATTLE, Dec. 13 Cell Therapeutics, Inc.,("CTI" or the ... of its new 5.75% Convertible Senior,Notes (the "New Notes") due 2011 and ...
Cached Biology Technology:BioMed Realty Trust Appoints Executive Officer of the Irvine Company to Its Board 2BioMed Realty Trust Appoints Executive Officer of the Irvine Company to Its Board 32007 a "Record Year" for Successful Validation Studies of Simbionix Simulators 2Cell Therapeutics, Inc. Announces Issuance of $23.25 Million in New Convertible Senior Notes in Exchange for $36.08 Million of Existing Convertible Senior Subordinated and Subordinated Notes 2Cell Therapeutics, Inc. Announces Issuance of $23.25 Million in New Convertible Senior Notes in Exchange for $36.08 Million of Existing Convertible Senior Subordinated and Subordinated Notes 3
(Date:10/1/2014)... TURKU, Finland , October 1, 2014 /PRNewswire/ ... breaking through in the life science field  ... detection technologies come and go over the years. ... golden standard. Photon upconversion is a novel detection ... during recent years. Now, finally, this technology is ...
(Date:9/30/2014)... is published today in the first authoritative scientific study ... on both sides of the grouse moor management debate. ... the Ecohydrology of River basins) project has shown that ... support red grouse populations for gun sports, has significant ... properties, river water chemistry and river ecology. , A ...
(Date:9/30/2014)... conducted by the Ocean Health Index rates the ... overall health. In addition, for the first time, ... ocean regions beyond national jurisdiction (high-seas areas) ... safeguarding biodiversity and providing sustainable food sources. , ... a partnership led by scientists from UC Santa ...
Breaking Biology News(10 mins):First-of-a-kind Solution for Utilizing Photon Upconversion 2Grouse moor burning causes widespread environmental changes 2Rating the planet's oceans 2Rating the planet's oceans 3
... For those living with gastrointestinal disorders, such as ulcers or ... or dietary changes. But what if one day treatment meant ... free of inflamed or diseased tissues? That is ... Institute of Technology (MIT) are hoping their new study findings ...
... The winners of NERC,s inaugural short article and photography ... London this evening. The winning essay, by Laura ... excitement she felt during a recent research voyage when ... black smoke across her monitoring screens. These are underwater ...
... the 20th International AIDS Conference (AIDS 2014) to be ... today announced 19 major plenary presentations, focusing on the primary ... sessions will open each day of the conference, which is ... 200 countries. "We are excited to have such ...
Cached Biology News:Manufacturing a new gut to treat GI diseases 2NERC announces the winner of its first photo and essay competition 2NERC announces the winner of its first photo and essay competition 320th International AIDS Conference (AIDS 2014) plenary speakers announced 220th International AIDS Conference (AIDS 2014) plenary speakers announced 3
... cDNA normalization is essential for hunting ... high-throughput approaches, such as EST sequencing ... normalization methods are not well suited ... The Evrogen TRIMMER kit, though, is ...
... Tecan has 20 years of expertise in ... and still offers the most powerful systems ... up to 40 000 in-microplate tests requiring ... integration of different modules (such as worktable ...
... 6 delivers enhanced statistics and statistical ... quality improvement managers. As an accepted ... 6 simplifies statistics for novice users ... by experts. It offers a powerful ...
Glycerol-based mounting medium for immunofluorescence microscopy. Formulated to retard photobleaching of fluorochromes such as fluorescein and rhodamine. Product can also be used as a diluent for fl...
Biology Products: