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T7 Express High Efficiency Sampler |
 | | | | T7 Express High Efficiency Sampler | | | |
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Prices are in US dollars and valid only for US orders.
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- Transformation efficiency: 0.6-1 x 109 cfu/µg pUC19 DNA
- T7 RNA Polymerase in the lac operon - no lambda prophage
- Tight control of expression by lacIq allow potentially toxic genes to be cloned
- Control of T7 RNA Polymerase by mutant lysozyme (LysY) allows toxic genes to be expressed
- LysY is a variant of T7 lysozyme lacking amidase activity, thus cells are less susceptible to lysis during induction
- Maintenance of lysozyme plasmid does not require antibiotic selection
- Deficient in proteases Lon and OmpT
- 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:
C3009I:
2 x 0.2 ml/tube of chemically competent T7 Expresst E. coli cells (C2566)
2 x 0.2 ml/tube of chemically competent T7 Express Iq E. coli cells (C3016)
2 x 0.2 ml/tube of chemically competent T7 Express lysY E. coli cells (C3010)
2 x 0.2 ml/tube of chemically competent T7 Express lysY/Iq E. coli cells (C3013) (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)
T7 Express Genotype: 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
T7 Express Iq Genotype: 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 [miniF-lacIq(CamR)]
T7 Express lysY Genotype: 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 [miniF-lysY (CamR)]
T7 Express lysY/Iq Genotype: 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 [miniF-lysY, lacIq(CamR)]
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, 10 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.
Transformation of a toxic mammalian clone into E. coli hosts. A T7 expression plasmid and the same plasmid containing a gene encoding a toxic mammalian protein were transformed into each host. Comparison of the relative transformation efficiencies demonstrates that the T7 Express hosts provide the levels of control necessary for transformation of potentially toxic clones. BL21(DE3) could not be transformed with the toxic clone.
T7-controlled expression of a non-toxic protein in E. coli hosts. A T7 expression plasmid containing a gene encoding an E. coli protein was transformed into each host, grown to 0.6 OD and induced for 3 hours. Comparison of soluble extracts from uninduced (-) and induced (+) cells shows superior control of expression in the T7 Express hosts while maintaining high levels of induced expression.
* 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
- Why are there no colonies or no growth in liquid culture (C3009)?
- Why is there no protein visible on gel or no activity (C3009)?
- Why is induced protein insoluble (C3009)?
- What are the solutions/recipes (C3009)?
- What are the strain properties (C3009)?
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 0.05 ml of Competent E. coli following the high efficiency protocol provided. 0.6-1 x 109 colonies formed/µg after an overnight incubation on LB-ampicillin plates at 37°C.
Untransformed T7 Express cells were also tested for resistance to phage Φ80, a standard test for resistance to phage T1, and sensitivity to ampicillin chloramphenicol, tetracycline, kanamycin and streptomycin.
Untransformed cells of all other strains were also tested for resistance to phage Φ80, a standard test for resistance to phage T1, resistance to chloramphenicol and sensitivity to ampicillin, tetracycline, kanamycin and streptomycin.
Companion Products
T7 Express Iq Competent E. coli (High Efficiency)
T7 Express lysY/Iq Competent E. coli (High Efficiency)
T7 Express lysY Competent E. coli (High Efficiency)
T7 Express Competent E. coli (High Efficiency)
T7 Express Crystal Competent E. coli (High Efficiency)
Legal
Research Use Assurance:
Notice to Buyer/User: The buyer and user have a nonexclusive license to use this system or any component thereof for RESEARCH PURPOSES ONLY. See Assurance Letter and Statement attached hereto for details on terms of the license granted hereunder.
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