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T7 Express Iq Competent E. coli |
 | | | | T7 Express Iq Competent E. coli | | | |
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This strain (C2833) has been discontinued and replaced by the NEB new high efficiency strain C3016.
- Transformation efficiency: 2 - 6 x 108 cfu/µg
- T7 RNA Polymerase in the lac operon - no lambda prophage
- Tight control of expression by lacIq allows potentially toxic genes to be cloned
- Deficient in proteases Lon and OmpT
- F´ allows cells to be infected with bacteriophage M13 for ssDNA production
- Resistant to phage T1 (fhuA2)
- Does not restrict methylated DNA (McrA-, McrBC-, EcoBr-m-, Mrr-)
- B Strain
Description:
Chemically competent E. coli cells suitable for high efficiency transformation and protein expression.
Reagents Supplied:
20 x 0.05 ml/tube of chemically competent NEB T7 Express Iq 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+ lacIqzzf::Tn10(TetR)/ fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) endA1 Δ(mcrC-mrr)114::IS10
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 T7 Express Iq Competent E. coli?
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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 T7 Express Iq Competent E. coli following the High Efficiency Protocol provided. Greater than 2 x 108 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.
Companion Products
dam-/dcm- Competent E. coli
NEB 10-beta Competent E. coli (High Efficiency)
NEB 5-alpha Competent E. coli (Subcloning Efficiency)
NEB 5-alpha F´Iq Competent E. coli (High Efficiency)
NEB Turbo Competent E. coli (High Efficiency)
T7 Express Competent E. coli (High Efficiency)
Legal
Patents:
New England Biolabs: U.S. Patent No. 6,569,699
Research Use Assurance:
The buyer and user have a non-exclusive sub-license to use this system or any component thereof for RESEARCH PURPOSES ONLY, based upon agreement to the following assurances.
Transfer of the host cells that contain the cloned copy of the T7 gene 1 to third parties is explicitly prohibited. This limitation applies to E. coli ER2566 and ER2833 and their competent derivatives, C2566 and C2833, when provided separately or when provided in combination with appropriate vectors for said systems.
Licenses/Patents/Disclaimers:
A license to use this system or any components thereof for commercial purposes may be obtained from New England Biolabs, Inc.
Commercial Laboratory Buyer and User:
Use of the host cells ER2566 or ER2833 or their competent derivatives that contain the cloned copy of T7 gene 1, the gene for T7 RNA polymerase for any purpose other than in combination with either a T7/MAL or T7/IMPACT vector is explicitly prohibited.
Use of host cells that may contain the cloned copy of the T7 gene 1, the gene for T7 RNA polymerase with any other vector(s) containing a T7 promoter to direct the production of RNA or protein requires a license from Brookhaven National Laboratory. Information about research-use or commercial-use license agreements may be obtained from the Office of Technology Transfer, Brookhaven National Laboratory, Building 475D, P.O. Box 5000, Upton, New York, 11973-5000; telephone: 631-344-7134; fax: 631-344-3729.
You may refuse this non-exclusive research license agreement by returning the enclosed materials unused. By keeping or using the enclosed materials, you agree to be bound by the terms of this sub-license.
ACADEMIC AND NON-PROFIT LABORATORY ASSURANCE LETTER
The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U.S. Department of Energy and is the subject of patent applications assigned to Brookhaven Science Associates, LLC (BSA). BSA will grant a non-exclusive license for use of this technology, including the enclosed materials, based upon the following assurances:
1. These materials are to be used for noncommercial research purposes only. A separate license is required for any commercial use, including the use of these materials for research purposes or production purposes by any commercial entity. Information about commercial licenses may be obtained from the Office of Intellectual Property and Industrial Partnerships, Brookhaven National Laboratory, Building 475D, P.O. Box 5000, Upton, New York 11973-5000, telephone (631) 344-7134.
2. No materials that contain the cloned copy of T7 gene 1, the gene for T7 RNA polymerase, may be distributed further to third parties outside of your laboratory, unless the recipient receives a copy of this license and agrees to be bound by its terms. This limitation applies to strains ER2566, ER2833, BL21(DE3), BL21(DE3)pLysS, and BL21(DE3)pLysE, their competent derivatives, and any derivatives you may make of them, including such strains containing recombinant vectors.
You may refuse this license by returning the enclosed materials unused. By keeping or using the enclosed materials, you agree to be bound by the terms of this license.
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