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NEB 5-alpha Competent E. coli (Subcloning Efficiency) |
 | | | | NEB 5-alpha Competent E. coli (Subcloning Efficiency) | | | |
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Prices are in US dollars and valid only for US orders.
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- Transformation efficiency > 1 x 106 cfu/µg
- Efficient transformation of unmethylated DNA derived from PCR, cDNA and many other sources (hsdR)
- 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
- Reduced recombination of cloned DNA (recA)
- K12 Strain
Description:
Chemically competent E. coli cells suitable for subcloning efficiency transformation in a wide variety of applications.
Genotype: fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17
Reagents Supplied:
6 x 0.4 ml/tube of chemically competent NEB 5-alpha Competent E. coli cells (Store at -80°C)
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.
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 QIAprep Spin 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 or phenol/chloroform extraction and ethanol precipitation should be added.
Effect of heat shock time on NEB 5-alpha competent E.coli (Subcloning Efficiency) 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 (Subcloning Efficiency) 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 (Subcloning Efficiency) 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 higher transformation efficiency than LB medium.
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 (Subcloning Efficiency)?
- What are the solutions/recipes that I need for transforming NEB 5-alpha Competent E. coli (Subcloning Efficiency)?
- What are the strain properties of NEB 5-alpha Competent E. coli (Subcloning Efficiency)?
- What is the difference between NEB #C2988J and NEB #C2987H?
- Can NEB 5-alpha competent E.coli (Subcloning Efficiency) (NEB #C2988J) be used for large fragment cloning?
- What is the optimal heat shock time for this strain (NEB #C2988J)?
- How long should I incubate cells on ice after DNA has been added (NEB #C2988J)?
- Can LB medium be used instead of SOC in the outgrowth step (C2988)?
Protocols
Protocols for NEB 5-alpha Competent E. coli (Subcloning Efficiency)
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Transformation Efficiency:
100 pg of pUC19 plasmid DNA was used to transform NEB 5-alpha Competent E. coli following the transformation protocol provided. Greater than 1 x 106 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 and sensitivity to ampicillin, chloramphenicol, kanamycin, streptomycin and tetracycline. The cells were shown to be suitable for blue/white screening by α-complementation of the β-galactosidase gene using pUC19.
Companion Products
NEB 5-alpha Competent E. coli (High Efficiency)
NEB 5-alpha Electrocompetent E. coli
NEB 5-alpha F´Iq Competent E. coli (High Efficiency)
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