Product Class: Other

NEB® Stable Competent E. coli (High Efficiency)

Supplied with outgrowth medium optimized for NEB 10-beta & NEB Stable Competent E.coli ; please do not use SOC outgrowth medium.

Product Introduction

Chemically competent E. coli cells suitable for high efficiency transformation.

  • Ideal for isolation of plasmid clones containing repeat elements and unstable inserts
  • Useful for isolating and propagating retroviral/lentiviral clones
  • Carries the endA mutation (in contrast to Stbl3) so that isolated plasmids are free of Endonuclease I
  • No dry ice surcharge on competent cell shipments
  • Outgrowth medium included
  • Free of animal products
Catalog # Size Concentration
C3040H 20.0 x 0.05 ml
C3040I 6.0 x 0.2 ml

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Product Information

Description

Highlights

  • 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 with vectors capable of alpha-complementation

Transformation Efficiency

1-3 x 109 cfu/µg pUC19 DNA

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

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)
This product is related to the following categories:
Cloning Competent Cell Strains Products
This product can be used in the following applications:
Transformation,
High-throughput cloning and automation solutions

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Antibiotic for Plasmid Selection

Antibiotics for Plasmid Selection Working Concentration
Ampicillin 100 µg/ml
Carbenicillin 100 µg/ml
Chloramphenicol 33 µg/ml
Kanamycin 30 µg/ml

Shipping Notes

  • Ships on dry ice

Antibiotic Resistance

  • tetracycline
  • streptomycin

Features

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

Application Features

  • Cloning unstable inserts
  • Isolating and propagating retroviral/ lentiviral clones
  • Compatible with NEBuilder HiFi DNA Assembly, 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.
Benefit from the high transformation efficiencies of NEB stable competent E. coli
NEBStable_Stbl3_TE_1217 Transformation efficiencies were compared using manufacturers’ recommended protocols. Values shown are the average of triplicate experiments.
Effect of outgrowth medium on transformation efficiency
C3040_OutgrowthMedium_0517a
50 μl of NEB Stable 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 10-beta/Stable Outgrowth Medium delivers the highest transformation efficiency.

Product 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.
  3. Transformation reactions with AmpR plasmids do not require an outgrowth period after addition of Outgrowth Medium. Plasmid selection using antibiotics other than ampicillin requires an outgrowth period of 60 minutes at 30°C before plating on selective media. 30°C or 37°C  may be used for plate incubation, however 30°C is recommended as some constructs may be unstable at elevated temperatures.

Protocols, Manuals & Usage

Protocols

  1. 5 Minute Transformation Protocol for NEB® Stable Competent E. coli (C3040H/C3040I)
  2. High Efficiency Transformation for NEB® Stable Competent E. coli (C3040H)
  3. Protocol for cloning DNA containing repeat elements (C3040)
  4. High Efficiency Transformation Protocol (C3040I)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Which competent cell strains are compatible with Gateway® Cloning?
  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 NEBuilder HiFi, Gibson Assembly or Golden Gate Assembly products?
  6. How should I calculate the transformation efficiency (C3040)?
  7. What is the optimal heat shock for this strain? (C3040)
  8. What volume of DNA can be added into competent cells?
  9. Is NEB Stable suitable for larger plasmid transformations?
  10. Is NEB Stable a substitute for Stbl2 or Stbl3?
  11. What type of competent cells are suitable for transformation of DNA constructs created using NEBuilder HiFi DNA Assembly Master Mix?
  12. How should I store the NEB 10-beta/Stable Outgrowth Medium?
  13. Do NEB Stable Competent E.coli require an outgrowth recovery period after transformation with AmpR plasmids?
  14. How should fragments be prepared for assembly using NEBuilder HiFi?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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