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NEB 5-alpha Competent E. coli |
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This product has been discontinued and replaced by NEB 5-alpha F'Iq Competent E. coli (High Efficiency)(#C2992)
- Transformation efficiency: 1 - 3 x 109 cfu/µg
- Efficient transformation of unmethylated DNA derived from PCR, cDNA and many other sources (hsdR)
- Tight control of expression by lacIq allows potentially toxic genes to be cloned
- Activity of nonspecific endonuclease I (endA) eliminated for highest quality plasmid preparations
- Resistance to phage T1 (fhuA2)
- Suitable for blue/white screening by α-complementation of the β-galactosidase gene
- F´ allows cells to be infected with bacteriophage M13 for ssDNA production
- Reduced recombination of cloned DNA (recA)
- K12 Strain
Description:
Chemically competent E. coli cells suitable for high efficiency transformation in a wide variety of applications.
Reagents Supplied:
20 x 0.05 ml/tube of chemically competent NEB 5-alpha Competent E. coli cells (Store at -80°C)
6 ml of SOC outgrowth medium (Store at room temperature)
0.025 ml of 50 pg/µl pUC19 Control DNA (Store at -20°C)
Genotype: F' proA+B+ lacIq (ΔlacZ)M15 zzf::Tn10 (TetR)/fhuA2Δ(argF-lacZ)U169 (Φ80 Δ(lacZ)M15) glnV44 gyrA96 (NalR) recA1 relA1 endA1 thi-1 hsdR17
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 you shorten this step.
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 you shorten this step. 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.
DNA Effects on Transformation Efficiency and Colony Output: The optimal amount of DNA to use in a transformation reaction is lower than commonly recognized. Using clean, supercoiled pUC19, the efficiency of transformation is highest in the 100 pg-1 ng range. However, the total colonies which can be obtained from a single transformation reaction increase up to about 100 ng.
* 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 (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.
Reaction & Storage Conditions
Storage Temperature:
-80°C
Notes
General notes:- 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.
FAQs
- How should I calculate the transformation efficiency of NEB 5-alpha Competent E. coli?
- What are the solutions/recipes that I need for transforming NEB 5-alpha Competent E. coli?
- What are the strain properties of NEB 5-alpha Competent E. coli from NEB?
Quality Control
Transformation Efficiency::
100 pg of pUC19 plasmid DNA was used to transform NEB 5-alpha Competent E. coli following the High Efficiency Protocol provided. Greater than 2 x 109 colonies formed/µg after an overnight incubation on LB-ampicillin plates at 37°C.
Untransformed cells were also tested for resistance to phage Φ80, a standard test for resistance to phage T1, resistance to tetracycline, and sensitivity to ampicillin, chloramphenicol, kanamycin and streptomycin. The cells were shown to be suitable for blue/white screening by α-complementation of the β-galactosidase gene using pUC19.
Quality control values for a specific lot can be found on the datacard which accompanies each product.
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