NEB® 10-beta Electrocompetent E. coli

Description

NEB 10-beta electrocompetent E. coli cells are optimized for high efficiency transformation by electroporation. These cells are ideal for DNA library constructions and all cloning purposes.

Highlights

  • DH10B™ derivative
  • Transformation efficiency: 2 x 1010 cfu/μg pUC19
  • Accomodation of large plasmids including BAC and cosmid constructs
  • Efficient transformation of methylated DNA derived from eukaryotic sources and unmethylated DNA derived from PCR, cDNA and other sources
  • Activity of nonspecific endonuclease I eliminated for highest quality plasmid preparations (endA1)
  • Suitable for blue/white screening by α-complementation of the β-galactosidase gene (Φ80ΔlacZM15)
  • Reduced recombination of cloned DNA (recA)
  • Resistance to phage T1 lytic (fhuA2)
  • K-12 Strain
  • Free of animal products

Genotype

Δ(ara-leu) 7697 araD139  fhuA ΔlacX74 galK16 galE15 e14-  Φ80dlacZΔM15  recA1 relA1 endA1 nupG  rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC) 

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
pUC19 Vector-200.05 ng/μl
NEB® 10-beta/Stable Outgrowth Medium4

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 than10-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. 
  8. Cold and dry selection plates lead to lower transformation efficiency. Pre-warm plates at 37°C for 1 hour. Using 37°C pre-warmed recovery medium increases the efficiency by about 20%. 
  9. Refreeze unused cells in a dry ice/ethanol bath for 5 min and then store at -80°C. Do not use liquid nitrogen. Additional freeze-thaw cycles result in lower transformation 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. Total colony output continues to increase with increasing DNA input up to at least 1 μg of puc19.

Properties and Usage

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

Storage Temperature

-80°C

Shipping Notes

  • Ships on dry ice

Antibiotic Resistance

  • str

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.

FAQs

  1. How should I calculate the Electrotransformation efficiency (C3020)?
  2. What are the strain properties (C3020)?
  3. What type of competent cells are suitable for transformation of DNA constructs created using Gibson Assembly?
  4. What type of competent cells are suitable for transformation of DNA constructs created using NEBuilder HiFi DNA Assembly Master Mix?
  5. How should I store SOC Outgrowth Medium? The SOC I received with my competent cells recommends storage at either room temperature or 4°C, however, when I purchase it as a stand alone product, it recommends storing it at 4°C. Which is better?
  6. How should I store the NEB 10-beta/Stable Outgrowth Medium?

Protocols

  1. Electroporation Protocol (C3020)

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.

Selection Charts

Usage Guidelines & Tips

Interactive Tools

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. Further information regarding NEB product quality can be found here.
  • 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.

Certificate of Analysis

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

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]

Safety Data Sheet

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

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.