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 (Str^R) rph spoT1 Δ(mrr-hsdRMS-mcrBC) 

Reagents Supplied

The following reagents are supplied with this product:

Advantages and Features

Features

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

Properties and Usage

Storage Temperature

Shipping Notes

• Ships on dry ice

Antibiotic Resistance

• str

Quality Control

Quality Control Assays

• 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.
   

Supporting Documents

Material Safety Datasheets

• NEB 10-beta Electrocompetent E. coli MSDS
• pUC19 Vector MSDS
• SOC Outgrowth Medium MSDS

Datacards

• C3020Datasheet-Lot40
• C3020Datasheet-Lot41
• C3020Datasheet-Lot44
• C3020Datasheet-Lot46
• C3020Datasheet-Lot50
• C3020Datasheet-Lot51
• C3020Datasheet-Lot52