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  • TransPass™ D2 Transfection Reagent

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    The TransPass D2 Transfection Reagent is a non-lipid cationic polymer designed for transfecting plasmid DNA into many mammalian cell lines including primary cell lines and endothelial cell lines. Complex co-transfections of many plasmids can be efficiently transfected using the TransPass D2 Transfection Reagent.


    Cell Lines Successfully Transfected:
    A549, CHO-K1, COS, HCT116, HEK293, HeLa, HepG2, HL60, Jurkat, MCF-7, MDA, NIH3T3 and U20S.

    Figure 1:
    Human Umbilical Vein Endothelial Cells (HUVEC) transfected with a plasmid expressing GFP using TransPass D2 Transfection Reagent (Pradhan et al., unpublished results).
    Figure 2:
    Titration of TransPass D2 Transfection Reagent in CHO-K1 cells using Protocol 1 and secreted Gaussia luciferase (pCMV-GLuc) as a reporter. Transfections contain 0.7 µg pCMV-GLuc and 250 µl plating medium (10%FBS-F12K) per well. The values obtained from the transfections with the indicated amounts of TransPass D2 Transfection Reagent were obtained from 20 µL of culture medium 24 hr post transfection.

    Properties and Usage

    Storage Temperature

    4°C

    Quality Control

    Quality Assurance Statement

    • Each lot of TransPass D2 Transfection Reagent is tested for efficient delivery of two different reporter plasmids expressing luciferase in CHO cells.

    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 D2 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. 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.
    4. 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).
    5. For suspension cells, the following protocol (modification of Protocol 1) is recommended: 
      Prepare the plasmid/TransPass D2 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.
    6. PLEASE NOTE:  This product cannot be frozen.

    References

    1. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning:  A Laboratory Manual (2nd ed.).
    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 D2 Protocol 1: Transfection in the presence of serum
    2. TransPass D2 Protocol 2: Transfection in the absence of serum

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