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  • TransPass™ D1 Transfection Reagent

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    The TransPass D1 Transfection Reagent is a formulation of cationic lipid-vesicles designed for transfecting plasmid DNA into many mammalian cell lines including primary endothelial cells. It is a frozen suspension that should be resuspended by vortexing before use. Complex co-transfections of many plasmids can be efficiently transfected using the TransPass D1 Transfection Reagent.

    Cell Lines Successfully Transfected:
    A549, COS, Drosophilia SL2, HEK293, HeLa, HO23, NIH3T3 and U20S.

    Primary Endothelial cells: HUVEC, HMVEC, lung microvascular endothelial cells, human aortic endothelial cells, bovine aortic endothelial cells.

    Figure 1:
    Primary human hepatocytes transfected with a plasmid expressing β-galactosidase using TransPass D1 Transfection Reagent.
    Figure 2:
    Cells transfected in 12 well plate format with 1.3 µg pCMVGLuc Control Plasmid and 3.9 µl TransPass D1 Transfection Reagent. Assays were performed in 20 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 must be optimized.

    Properties and Usage

    Storage Temperature

    -20°C

    Quality Control

    Quality Assurance Statement

    • Each lot of TransPass D1 Transfection Reagent is tested for efficient delivery of two different plasmids in different cell lines.

    Notes

    1. For most cell lines try Protocol 1 first (transfection in the presence of serum). Keep the amount of plasmid DNA constant while varying the amount of TransPass D1 Transfection Reagent. Once the optimal ratio (transfection reagent to DNA) is established, the amount of plasmid DNA and transfection reagent can be increased. Use Table 1 as a guide to optimize the transfection protocol for different plate formats. 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.
    2. For both Protocols 1 and 2, serum-free medium is required to form the transfection complexes (Step 2).
    3. The TransPass D1 Transfection Reagent tube should be vortexed just before use to thoroughly mix the suspension.
    4. Multiple plasmids can be combined (step 2) and simultaneously transfected. For consistent results, the total amount of transfected DNA should stay the same between different wells.
    5. If you are using Protocol 2 and the cells are sensitive to prolonged absence of serum, the exposure to the serum-free medium can be minimized by decreasing the incubation time (Step 7).
    6. For suspension cells, the following protocol (modification of Protocol 1) is recommended: 
      Prepare the plasmid/TransPass D1 complex in 0.5 ml serum-free medium per well of a 12-well plate. Incubate at room temperature for 20-30 minutes. Immediately prior to adding transfection complex (step 5 of Protocol 1), spin down cells to be transfected and resuspend them in high glucose DMEM at a density of 5 x 106 cells per ml. Aliquot 100 µl of cell suspension per well of a 12-well plate and add 0.5 ml of the transfection complex. Rock the plate gently in order to evenly disperse the complex mixture and incubate the cells for two hours. Add complete media with serum as follows: 2 ml per well for a 6-well plate, 1 ml per well for a 12-well plate and 0.5 ml per well for a 24-well plate.

    References

    1. Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, (2nd ed.). 1989
    2. Ausubel, F. M. et al. (1987). Current Protocols in Molecular Biology, (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 D1 Protocol 1: Transfection in the presence of serum
    2. TransPass D1 Protocol 2: Transfection in the absence of serum

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