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
Transformation efficiency: >1 x 1010 cfu/µg pUC19 DNA
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 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.
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.
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.
CAUTION: This product contains DMSO, a hazardous material. Review the MSDS before handling.
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.)
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
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.
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]
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]
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.)
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at gbd@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.
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