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Breeding medium for preparing cells in competence of electric transform in high effectively, and application

A technology for competent cells and cell culture, applied in the field of electrotransformation competent cells, can solve the problems of high price and high price, and achieve the effects of simple preparation, low cost and reduced material cost.

Inactive Publication Date: 2007-10-10
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

Invitrogen, Stratagene, Lucigen and many other well-known foreign biological reagent companies have high-quality electrotransformation competent cells for sale, but the price is expensive
Take Invitrogen ElectroMAX DH10B Competent Cell as an example, although its conversion efficiency can reach 1×10 10 cfu / μg pUC19 DNA, but the price is as high as $258 per 500μl. For a BAC library with an average insert size of 150kb and 150,000 clones, the cost of competent cells alone is nearly $30,000

Method used

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  • Breeding medium for preparing cells in competence of electric transform in high effectively, and application

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Embodiment

[0022] 1. Preparation of YENB medium: For each liter of medium, add 8g of yeast extract and 8g of nutrient broth to 950ml of ultrapure water, shake the container until the solute is completely dissolved. Adjust the pH to 7.0 with 5mol / L NaOH, and dilute to 1L with ultrapure water. At 15psi (1.05kg / cm 2 ) steam sterilization under high pressure for 20min.

[0023] 2. Selection of culture medium:

[0024] Using YENB and SOB (Mg-free) with different nutritional components and ionic strength 2+ ), 2×YT, LB medium for selection and optimization of the medium (SOB, 2×YT, LB formulations refer to the third edition of Molecular Cloning);

[0025] The culture conditions of DH10B competent cells are as follows: take out the DH10B strain from the ultra-low temperature refrigerator, streak on the non-resistant LB solid medium plate, and culture it upside down at 37°C for 14-16 hours. Pick individual round and glossy single colonies and inoculate 10ml YENB culture-based 50ml conical-bo...

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Abstract

This invention relates to a YENB culture medium for preparing high-efficiency electroporation-competent cells. The YENB culture medium comprises (per liter): yeast extract for cell culture 6-9 g, nutrient broth for cell culture 6-9 g, and water. The YENB culture medium can prepare high-efficiency electroporation-competent cells by controlling cell primary and secondary growth time, centrifugal speed and time. The conversion rate is 2.19X1010 cfu / mu.g pUC19 DNA, which is higher than imported culture medium, while the cost is only 5% that of imported culture medium. The culture medium lays a basis for high-efficiency conversion of large fragment DNA genomic library.

Description

technical field [0001] The present invention relates to electrotransformation competent cell, specifically a kind of medium for preparing competent cell and its application, using this special medium to cultivate Gram-negative bacteria—Escherichia coli (Escherichia coli, E.coli) DH10B strain was used to prepare high-efficiency electroporation competent cells. Background technique [0002] Transformation is a key technique in molecular cloning to introduce foreign DNA into recipient cells. Since Mandle and Higa discovered CaCl in 1970 2 After the treated cells could be transfected with lambda phage DNA, in 1972 and 1973, Cohen et al. used the same method to successfully introduce R factor and recombinant plasmid DNA into E. coli for the first time, thus establishing the genetic transformation technology. There are many conversion methods, such as calcium chloride method, Hanahan method and Inoue method, etc., which can meet the general experimental needs, but the conversion...

Claims

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Application Information

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IPC IPC(8): C12N1/00C12N1/20C12R1/19
Inventor 相建海张洋刘斌张晓军李富花
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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