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  • TransPass™ COS/293 Transfection Reagent

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    The TransPass™ COS/293 Transfection Reagent is TransPass™ D2 Transfection Reagent, a non-lipid cationic polymer, tested for the transfection of plasmid DNA into COS and HEK293 cell lines in a serum-compatible protocol. Complex co-transfections of many plasmids can be efficiently transfected using the TransPass COS/293 Transfection Reagent.

    Figure 1:
    COS-7 cells grown and transfected on a slide coverslip with GFP-expressing plasmid and 2 µl TransPass COS/293 Transfection Reagent in 6-well plate format. Data shown is 24 hr post-transfection (Pradhan et al., unpublished results).
    Figure 2:
    Titration of TransPass COS/293 Transfection Reagent in HEK293 cells using secreted Gaussia Luciferase (pCMV-GLuc Control Plasmid) as a reporter. Cells are transfected in 12-well plate format and GLuc activity was measured in 10 µl cell supernatant 24 hr post-transfection.

    Advantages and Features

    Applications


    Background:

    The introduction of recombinant DNA into cultured cells or "transfection", has become an essential tool for studying gene function and regulation. Initially, cells were transfected by methods such as Calcium Phosphate co-precipitation, electroporation or viral factors (1, 2). The introduction of cationic lipids or polymers as transfection agents has led to the development of reagents that efficiently deliver DNA through the cell membrane and into the nucleus (3).

    Transfection Guidelines:
    • For consistent results, it is important to maintain healthy proliferating cells that are regularly passaged. 
    • It is important that NO heparin and NO antibiotics/antimycotics in the growth medium during transfection.
    • Use sterile plasmid DNA that is purified by CsCl gradient centrifugation or column chromatography. 
    • The following parameters can be optimized in order to maximize the transfection efficiency for a particular cell line: the cell density at the time of transfection, amount of transfection reagent, amount of plasmid DNA and the culture incubation time before analysis. The specific amount of transfection reagent can be optimized for a particular cell line by performing a simple titration (Figure 2).

    Properties and Usage

    Storage Temperature

    4°C

    Quality Control

    Quality Assurance Statement

    • Each lot of transfection reagent is tested for efficient delivery of two different reporter plasmids in COS-7 and HEK293 cells.

    Notes

    1. In order to form the transfection complexes, serum-free medium is required.
    2. For optimal transfection of a certain cell density the amount of TransPass COS/293 Transfection Reagent can be titrated while keeping the amount of plasmid DNA constant. Use Table 1 as a guideline for amounts using different plate sizes. A convenient method for optimizing transfection conditions is to use pCMV-GLuc 2 Control Plasmid (NEB #N8081), which contains the gene for the secreted Gaussia Luciferase. See Figure 2 for an example titration in HEK293 cells. In addition, multiple plasmids can be combined and simultaneously transfected.
    3. PLEASE NOTE:  This product cannot be frozen.

    References

    1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: Cold Spring Harbor Laboratory . (2nd ed.)
    2. Ausubel, F. M. et al. (1987). Current Protocols in Molecular Biology, New York: Greene Publishing Associates and Wiley- . (2nd ed.)
    3. Felgner P.L. et al. (1987). Proc. Natl. Acad. Sci. USA. 84, 7413-7417.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Cellular Imaging and Analysis FAQs
    1. TransPass COS/293: Transfection Protocol

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