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  • NEB 5-alpha Competent E. coli (Subcloning Efficiency)

    Description

    Chemically competent E. coli cells suitable for subcloning efficiency transformation in a wide variety of applications.

    Highlights

    • Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations
    • Transformation efficiency > 1 x 106 cfu/µg pUC19 DNA
    • Efficient transformation of unmethylated DNA derived from PCR, cDNA and many other sources (hsdR)
    • Reduced recombination of cloned DNA (recA1)
    • Resistance to phage T1 (fhuA2)
    • Suitable for blue/white screening by α-complementation of the β-galactosidase gene
    • K12 Strain
    • DH5α™ derivative
    • Free of animal products

    Genotype

    fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17

    Advantages and Features

    Features

    Transformation Protocol Variables
    Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.

    Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.

    Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.

    Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.

    Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.

    Applications


    Effect of DNA Purity on Transformation Efficiency and Colony Output: The total colonies which can be obtained from a single transformation reaction with NEB 5-alpha Competent E.coli (Subcloning Efficiency) are not significantly reduced when using miniprep DNA. Using 10 ng-1000 ng of clean, supercoiled pUC19 or pUC19 isolated with a QIAprep Spin Miniprep Kit, total colonies increase with increasing DNA concentration.
    * Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants, a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.
    Effect of heat shock time on NEB 5-alpha competent E.coli transformation efficiency: 50 μl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except heat shock time varied from 0 to 80 seconds.
    Effect of DNA incubation time on NEB 5-alpha competent E.coli transformation efficiency: 50 μl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except DNA incubation time varied from 0 to 40 minutes.
    Effect of outgrowth medium on transformation efficiency: 50 μl of NEB 5-alpha competent E.coli was transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol with the exception of varying the outgrowth medium. NEB SOC outgrowth medium delivers the highest transformation efficiency.

    Properties and Usage

    Antibiotics for Plasmid SelectionWorking Concentration
    Ampicillin100 μg/ml
    Carbenicillin100 μg/ml
    Chloramphenicol33 μg/ml
    Kanamycin30 μg/ml
    Streptomycin25 μg/ml
    Tetracycline15 μg/ml

    Storage Temperature

    -80°C

    Shipping Notes

    • Ships on dry ice

    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.

    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. Can LB medium be used instead of SOC in the outgrowth step (C2988)?
    2. Can NEB 5-alpha competent E.coli (Subcloning Efficiency) (NEB #C2988J) be used for large fragment cloning?
    3. What is the difference between NEB #C2988J and NEB #C2987H?
    4. What is the optimal heat shock time for this strain (NEB #C2988J)?
    5. How long should I incubate cells on ice after DNA has been added (NEB #C2988J)?
    6. How should I calculate the transformation efficiency of NEB 5-alpha Competent E. coli (Subcloning Efficiency)?
    7. What are the solutions/recipes that I need for transforming NEB 5-alpha Competent E. coli (Subcloning Efficiency)?
    8. What are the strain properties of NEB 5-alpha Competent E. coli (Subcloning Efficiency)?
    9. Can I store competent cells at -20°C instead of -80°C?
    10. Which kind of transformation tubes should be used?
    11. What volume of DNA can be added into competent cells?
    12. What is the shelf life for this strain (NEB #C2988J)?
    13. Are NEB's competent cells compatible with the "Plate and Go" protocol?
    1. Transformation Protocol (C2988)
    2. 5 Minute Transformation Protocol (C2988)

    Selection Tools

    Usage Guidelines & Tips

    Application Notes