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NEB 10-beta Electrocompetent E. coli |
 | | | | NEB 10-beta Electrocompetent E. coli | | | |
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Prices are in US dollars and valid only for US orders.
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- K-12 Strain
- Clone large plasmids and BACs
- DH10B™ derivative
- Fast growing recA strain
- Free of animal products
- T1 phage resistant (fhuA2)
Description:
NEB 10-beta electrocompetent E. coli cells are optimized for high efficiency transformation by electroporation. These cells are ideal for DNA library constructions and all cloning purposes.
Reagents Supplied:
C3020K:
6 x 0.1 ml/tube of NEB 10-beta Electrocompetent E. coli cells (Store at -80°C)
25 ml of SOC outgrowth medium (Store at room temperature)
0.025 ml of 50 pg/µl pUC19 Control DNA (Store at -20°C)
Genotype: araD139 Δ(ara-leu)7697 fhuA lacX74 galK (Φ80 Δ(lacZ)M15) mcrA galU recA1 endA1 nupG rpsL Δ(mrr-hsdRMS-mcrBC)Electroporation Tips: - 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 than10-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 are recommended for clean up 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.
- Cold and dry selection plates lead to lower transformation efficiency. Pre-warm plates at 37°C for 1 hour. Using 37°C pre-warmed recovery medium increases the efficiency by about 20%.
- Refreeze unused cells in a dry ice/ethanol bath for 5 min and then store at -80°C. Do not use liquid nitrogen. Additional freeze-thaw cycles result in lower transformation efficiency.
DNA Effects on Transformation Efficiency and Colony Output: Electrotransformation efficiency remains extremely high up to about 10 ng of input DNA, then decreases precipitously at higher DNA concentrations. Total colony output continues to increase with increasing DNA input up to at least 1 μg.
Advantages:- Transformation efficiency: 2-4 x 1010 cfu/μg pUC19
- Accomodation of large plasmids including BAC and cosmid constructs
- Efficient transformation of methylated DNA derived from eukaryotic sources and unmethylated DNA derived from PCR, cDNA and other sources
- Activity of nonspecific endonuclease I eliminated for highest quality plasmid preparations (endA1)
- Suitable for blue/white screening by α-complementation of the β-galactosidase gene (Φ80ΔlacZM15)
- Reduced recombination of cloned DNA (recA)
- Resistance to T1 lytic phage (fhuA2)
Reaction & Storage Conditions
Storage Temperature:
-80°C
Notes
General notes:- 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 Electrotransformation efficiency (C3020)?
- What are the solutions/recipes (C3020)?
- What are the strain properties (C3020)?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Transformation Efficiency:
10 pg of pUC19 plasmid DNA was electroporated into 25 µl NEB 10-beta Electrocompetent E. coli following the protocol described below. 1-3 x 1010 colonies formed/µg pUC19 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 streptomycin, and sensitivity to ampicillin, chloramphenicol, kanamycin 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 10-beta Competent E. coli (High Efficiency)
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