Preparing method for electrically transforming competent cells without buffer solution

A technology of competent cells and electrotransformation, applied in the field of bacterial molecular genetics, can solve the problems of high cost and cumbersome buffer preparation, and achieve the effect of improving the conversion rate

Pending Publication Date: 2018-09-28
SHAANXI SCI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, for the competent cells prepared by conventional methods, the preparation process requires a specific buffer, such as the conventionally selected buffer composition: the TB transformation buffer of every 20mL is composed of the following components: 12.5mL of double distilled water, 4mL of 1mol / L of KCl, 2.4mL of 0.45mol / L MnCl 2 , 0.5mol / L of CaCl 2 0.6mL and 0.5mL of 0.5mol / L K-MES buffer, the buffer is not only cumbersome to prepare, but also expensive

Method used

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Examples

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Effect test

preparation example Construction

[0027] The invention provides a preparation method for electrotransformation competent cells without buffer solution, comprising the following steps:

[0028] 1) Escherichia coli is inoculated in LB liquid medium containing magnesium ions for the first culture to obtain the first culture;

[0029] 2) Mixing the first culture obtained in the step 1) with the LB liquid medium, and performing the second culture when the OD600 value is 0.08-0.10, and obtaining the second culture when the OD600 value is 0.35-0.40;

[0030] 3) performing the first centrifugation on the second culture obtained in step 2), and discarding the supernatant to obtain the first precipitate; the time for the first centrifugation is 15 to 25 minutes;

[0031] 4) Mix the first precipitate obtained in step 3) with water, perform second centrifugal separation after the first precipitate is suspended, and discard the supernatant to obtain the second precipitate; the second centrifugal separation The time is 10-...

Embodiment 1

[0070] 1. Activate Escherichia coli using the conventional method of activating Escherichia coli, pick a single colony of activated Escherichia coli into 50ml LB liquid medium, and culture it with shaking at 37°C and 250rpm for 14 hours to obtain the first culture;

[0071] 2. Add the first culture to OD in 200ml LB 600 When the value is 0.12, carry out the second culture at 18°C ​​and 250rmp until the OD 600 When being 0.5, obtain the second culture;

[0072] 3. Pour the second culture into a pre-cooled sterile tube, and centrifuge at 5000rpm for 20min at 0°C to precipitate colonies;

[0073] 4. Discard the supernatant, add sterilized and pre-cooled water, suspend all the colonies, finally add water to 200ml, and centrifuge at 5000rpm for 15min at 0°C;

[0074] 5. Discard the supernatant, add sterilized and pre-cooled water to suspend all the colonies, finally add water to 100ml, and centrifuge at 4000rpm for 10min at 0°C;

[0075] 6. Repeat step 5 once;

[0076] 7. Disca...

Embodiment 2

[0080] 1. Activate Escherichia coli using the conventional method of activating Escherichia coli, pick a single colony of activated Escherichia coli and put it into 50ml of LB liquid medium containing magnesium ions, the concentration of magnesium ions is 0.01M, shake and culture at 37°C and 250rpm for 14 hours , to obtain the first culture;

[0081] 2. Add the first culture to OD in 200ml LB 600 When the value is 0.08, carry out the second culture at 18°C ​​and 250rmp until the OD 600 When being 0.35, obtain the second culture;

[0082] 3. Pour the second culture into a pre-cooled sterile tube, and centrifuge at 5000rpm for 20min at 0°C to precipitate colonies;

[0083] 4. Discard the supernatant, add sterilized and pre-cooled water, suspend all the colonies, finally add water to 200ml, and centrifuge at 5000rpm for 15min at 0°C;

[0084] 5. Discard the supernatant, add sterilized and pre-cooled water to suspend all the colonies, finally add water to 100ml, and centrifuge ...

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Abstract

The invention provides a preparing method for electrically transforming competent cells without a buffer solution, and belongs to the technical field of molekulare bakteriengenetik. The preparing method comprises the following steps that colibacillus is inoculated into a liquid culture medium containing magnesium ions for first culture; the first culture is mixed with the liquid culture medium, and then secondary culture is conducted to obtain a second culture, the second culture is subjected to centrifugal separation, and precipitation is obtained; the precipitation is mixed with water, afterthe precipitation is suspended, centrifugal separation is conducted, and precipitation is obtained; the precipitation is mixed with water, after the precipitation is suspended, centrifugal separationis conducted, precipitation is obtained, the operation is repeated one time, and precipitation is obtained; the precipitation is mixed with water, after the precipitation is suspended, centrifugal separation is conducted, and precipitation is obtained; the precipitation is mixed with water, after being suspended, the precipitation is evenly mixed with dimethyl sulfoxide, and electrically-transformed competent cells are obtained. By means of the preparing method, the high- transformation-rate competent cells can be obtained, the operation is simple, and the preparation cost is low.

Description

technical field [0001] The invention belongs to the technical field of bacterial molecular genetics, and in particular relates to a preparation method of electrotransformation competent cells without buffer solution. Background technique [0002] Escherichia coli can absorb exogenous DNA after being treated. Cells in this state are called "competent cells". The preparation of competent cells is an important link in molecular biology research, and the transformation efficiency directly affects the subsequent Experimental work carried out. Commonly used methods for preparing competent cells include CaCl 2 method, Hanahan and Inoue method. At present, the competent cells prepared by conventional methods require specific buffers in the preparation process, such as conventionally selected buffer components: the TB conversion buffer of every 20mL is composed of the following components: 12.5mL of double distilled water, 4mL of 1mol / L of KCl, 2.4mL of 0.45mol / L MnCl 2 , 0.5mol...

Claims

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Application Information

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IPC IPC(8): C12N1/36C12N1/20C12R1/19
CPCC12N1/20C12N1/36
Inventor 曲东燕飞
Owner SHAANXI SCI TECH UNIV
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