This product has been discontinued. Alternatives are NEB® 10-beta Electrocompetent E. coli (NEB #C3020K) or chemically competent cells, NEB® 5-alpha Competent E. coli (High Efficiency) (NEB #C2987)
Catalog # C2989 was discontinued on August 02, 2021
NEB 5-alpha Electrocompetent Competent E. coli is a derivative of the popular DH5α. It is T1 phage resistant and endA deficient for high-quality plasmid preparations.
Optimized for high efficiency transformation by electroporation
NEB 5-alpha Electrocompetent E. coli cells are optimized for high transformation efficiency by electroporation. These cells are ideal for DNA library constructions and all cloning purposes.
Highlights
DH5α™ derivative
Efficient transformation of unmethylated DNA derived from PCR, cDNA and many other sources (hsdR)
Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations
Suitable for blue/white screening by α-complementation of the β-galactosidase gene (Φ80Δ lacZM15)
Electroporation cuvettes and microcentrifuge tubes should be pre-chilled on ice.
Electrocompetent cells should be thawed on ice and suspended well by carefully flicking the tubes.
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.
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 than 10-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 (NEB #T1030) are recommended for cleanup of ligation reactions.
Electroporation conditions vary with different cuvettes and electroporators. If you are using electroporators or cuvettes 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.
Arcing may occur due to high concentration of salts or air bubbles.
It is essential to add recovery medium to the cells immediately after electroporation. One minute delay can cause a 3-fold reduction in efficiency.
Application Features
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.
CAUTION: This product contains DMSO, a hazardous material. Review the MSDS before handling.
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.
Singh A, Minia I, Droll D, Fadda A, Clayton C, Erben E (2014) Trypanosome MKT1 and the RNA-binding protein ZC3H11: interactions and potential roles in post-transcriptional regulatory networks Nucleic Acids Res; 42(7), 4652-68. PubMedID: 24470144, DOI: 10.1093/nar/gkt1416
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