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  • NEB 5-alpha Electrocompetent E. coli

    Description

    NEB 5-alpha Electrocompetent E. coli cells are optimized for high transformation efficiency by electroporation. These cells are ideal for DNA library constructions and all cloning purposes.

    Highlights

    • DH5α™ derivative
    • Transformation efficiency: >1 x 1010 cfu/µg pUC19 DNA
    • Efficient transformation of unmethylated DNA derived from PCR, cDNA and many other sources (hsdR)
    • Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations
    • Suitable for blue/white screening by α-complementation of the β-galactosidase gene (Φ80Δ lacZM15)
    • Resistance to phage T1 (fhuA2)
    • Reduced recombination of cloned DNA (recA1)
    • K12 Strain
    • Free of animal products

    Genotype

    fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pUC19 Transformation Control Plasmid-200.05 ng/μl
    SOC Outgrowth Medium41X

    Advantages and Features

    Features

    Electroporation Tips
    1. Electroporation cuvettes and microcentrifuge tubes should be pre-chilled on ice. 
    2. Electrocompetent cells should be thawed on ice and suspended well by carefully flicking the tubes. 
    3. Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells. The maximum recommended volume of a DNA solution to be added is 2.5 µl. Addition of a large volume of DNA decreases transformation efficiency. 
    4. Contaminants such as salts and proteins can lower electroporation efficiency. Ideally, DNA for transformation should be purified and suspended in water or TE. Transformation efficiency is more than 10-fold lower for ligation mixtures than the control pUC19 plasmid due to the presence of ligase and salts. If used directly, ligation reactions should be heat-inactivated at 65°C for 20 min and then diluted 10-fold. For optimal results, spin columns are recommended for clean up of ligation reactions.
    5. Electroporation conditions vary with different cuvettes and electroporator. If you are using electroporators not specified in the protocol, you may need to optimize the electroporation conditions. Cuvettes with 1mm gap are recommended (e.g. BTX Model 610/613 and Bio-Rad #165-2089). Higher voltage is required for cuvettes with 2 mm gap. 
    6. Arcing may occur due to high concentration of salts or air bubbles.
    7. It is essential to add recovery medium to the cells immediately after electroporation. One minute delay can cause a 3-fold reduction in efficiency.

    Applications


    DNA Effects on Transformation Efficiency and Colony Output: Electroporation efficiency remains extremely high up to about 10 ng of input DNA, then decreases at higher DNA concentrations.

    Properties and Usage

    Antibiotics for Plasmid SelectionWorking Concentration
    Ampicillin100 μg/ml
    Carbenicillin100 μg/ml
    Kanamycin30 μg/ml
    Streptomycin25 μg/ml
    Tetracycline15 μg/ml

    Storage Temperature

    -80°C

    Shipping Notes

    • Ships on dry ice

    Antibiotic Resistance

    • none

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Transformation Efficiency:
      The competent cells are tested for transformation efficiency and pass minimum release criteria. Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 μg of plasmid into a given volume of competent cells.

    Notes

    1. CAUTION: This product contains DMSO, a hazardous material. Review the MSDS before handling.
    2. STORAGE AND HANDLING: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.

    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. How should I calculate the Electrotransformation efficiency (C2989)?
    2. What are the solutions/recipes (C2989)?
    3. What are the strain properties (C2989)?
    1. Electroporation Protocol (C2989)

    Selection Tools

    Usage Guidelines & Tips