Chemically competent E. coli cells suitable for subcloning efficiency
transformations, such as plasmid transformation or routine subcloning
(e.g,. inserting a 1 kb fragment into a 2 kb vector). The efficiency of
these cells is not sufficiently high for most other cloning
applications. For that, we recommend NEB® 5-alpha Competent E. coli
(High Efficiency) (NEB #C2987) which has 1,000-fold higher
transformation efficiency.
Highlights
Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations
Efficient transformation of unmethylated DNA derived from PCR, cDNA and many other sources (hsdR)
Reduced recombination of cloned DNA (recA1)
Resistance to phage T1 (fhuA2)
Suitable for blue/white screening by α-complementation of the β-galactosidase gene
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
C2988J
-80
NEB® 5-alpha Competent E. coli (Subcloning Efficiency)
C2988JVIAL
-80
6 x 0.4 ml
Not Applicable
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
Streptomycin
25 µg/ml
Tetracycline
15 µg/ml
Shipping Notes
Ships on dry ice
Advantages and Features
Features
Transformation Protocol Variables Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.
Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.
Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.
Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.
Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.
Application Features
Effect of DNA Purity on Transformation Efficiency and Colony Output: The total colonies which can be obtained from a single transformation reaction with NEB 5-alpha Competent E.coli (Subcloning Efficiency) are not significantly reduced when using miniprep DNA. Using 10 ng-1000 ng of clean, supercoiled pUC19 or pUC19 isolated with a commercial miniprep kit, total colonies increase with increasing DNA concentration.
* Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants, a purification step, either a spin column (NEB #T1030) or phenol/chloroform extraction and ethanol precipitation should be added.
Effect of heat shock time on NEB 5-alpha competent E.coli transformation efficiency: 50 μl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except heat shock time varied from 0 to 80 seconds.
Effect of DNA incubation time on NEB 5-alpha competent E.coli transformation efficiency: 50 μl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except DNA incubation time varied from 0 to 40 minutes.
Effect of outgrowth medium on transformation efficiency: 50 μl of NEB 5-alpha 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 SOC outgrowth medium delivers the highest transformation efficiency.
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 & Change Notifications
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]
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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.
