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  • NEB Stable Competent E. coli (High Efficiency)

    Description

    Chemically competent E. coli cells suitable for high efficiency transformation and isolation of plasmid clones containing repeat elements.

    Highlights

    • Transformation efficiency: 1-5 x 108 cfu/µg pUC19 DNA
    • Recommended host strain for cloning genes into retroviral/ lentiviral vectors 
    • Recommended for cloning of direct repeats and inverted repeat sequences
    • Reduced recombination of cloned DNA (recA1)
    • Efficient transformation of methylated DNA derived from eukaryotic sources or unmethylated DNA derived from PCR, cDNA and many other sources [mcrAΔ(mrr-hsdRMS-mcrBC)]
    • Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations
    • Resistance to phage T1 (fhuA)
    • Suitable for blue/white screening without IPTG by alpha--complementation of the beta-galactosidase gene

    Genotype

    F' proA+B+ lacIq ∆(lacZ)M15 zzf::Tn10 (TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14-  Φ80dlacZ∆M15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 ∆(mrr-hsdRMS-mcrBC)

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Advantages and Features

    Features

    • High quality plasmid preparations due to endA mutation
    • Rapid growth recA strain
    • T1 phage resistance (fhuA)
    • Value pricing
    • Free of animal products

    Applications

    • Cloning unstable inserts
    • Isolating and propagating retroviral/ lentiviral clones
    • Compatible with Gibson Assembly reactions as well as ligation reactions

    NEB Stable enables the isolation of plasmid clones containing repetitive DNA elements:


    Plasmid pUC-5xREP contains five 32-bp repeats, making it unstable in a recombination-proficient strain. A) NEB Stable competent cells or B) Stbl3 competent cells were transformed with 2 µl of a pUC-5xREP Gibson Assembly reaction containing 2.2 ng (0.00125 pmol) pUC19 vector and approximately 1 ng (0.0028 pmol) 5xREP insert. Transformed cultures were plated on LB plates containing 100 µg/ml ampicillin and incubated overnight at 30°C. The next day, colony PCR was performed using M13/pUC polylinker primers to analyze 5xREP insert stability. Figure 1 shows the results of analyzing 33 independent colonies. The correct full-length amplicon is 623bp. 

    Properties and Usage

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

    Storage Temperature

    -80°C

    Shipping Notes

    • Ships on dry ice

    Antibiotic Resistance

    • tetracycline
    • streptomycin

    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. 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.
    2. CAUTION: This product contains DMSO, a hazardous material. Review the MSDS before handling.

    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.
    1. What are the solutions/ recipes for using NEB Stable Competent E. coli (High Efficiency) (C3040)?
    2. What are the strain properties (C3040)?
    3. What is the shelf-life for this strain?
    4. Which strain of Competent E.coli should I use for general cloning?
    5. Are NEB Stable Competent E. coli cells suitable for transformation of large plasmids and large Gibson Assembly products?
    6. How should I calculate the transformation efficiency?
    7. What is the optimal heat shock for this strain? (C3040)
    8. What volume of DNA can be added into chemically competent cells?
    9. Is NEB Stable suitable for larger plasmid transformations?
    10. Is NEB Stable a suitable substitute for chemical competent Stbl2 or Stbl3?
    1. 5 Minute Transformation Protocol (C3040)
    2. High Efficiency Plasmid Transformation Protocol (C3040)
    3. Protocol for cloning DNA containing repeat elements (C3040)

    Selection Tools

    Usage Guidelines & Tips