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Preparation method of competent cells with high transformation efficiency

A technology of competent cells and transformation efficiency, which is applied in the field of preparation of competent cells with high transformation efficiency, can solve the problems of low transformation rate and yield, inconvenient operation, etc., and achieve high transformation efficiency, easy control, and high yield.

Active Publication Date: 2016-10-26
XIAMEN LIFEINT TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of inconvenient operation, low conversion rate and yield in the prior art, the present invention provides a method for preparing competent cells with high conversion efficiency

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] (1) Streak a small amount of Escherichia coli TG1 strains stored at -80°C on an LB plate and culture overnight at 37°C;

[0021] (2) Pick a single clone and culture it in a 1L Erlenmeyer flask containing 250mL SOC medium, and culture it to OD at 18°C ​​and 250rpm 600 The value is between 2.0-3.0; where the cell growth density is 5×10 cells per milliliter of culture medium 7 About one is better, that is, the bacteria in the logarithmic phase or the early logarithmic growth period can be measured by measuring the OD of the culture medium. 600 value to control;

[0022] The preparation steps of SOC medium are as follows: add 20g peptone, 5g yeast extract, 0.5g NaCl, 0.186g KCl into 900mL ultrapure water, stir well to dissolve, adjust the pH to 7.0 with 1M HCl, and use ultrapure water to make up the volume to 1L, after autoclaving, add 5mL of sterilized 2M MgCl 2 , add 20mL filter-sterilized 1M Glucose, mix well, and store at 4°C;

[0023] (3) Transfer the culture to a ...

Embodiment 2

[0030] (1) Take a small amount of bacterial strains and streak on an LB plate without resistance, and culture overnight at 37°C;

[0031] (2) Pick a single clone and culture it in a 1L Erlenmeyer flask containing 250mL SOC medium, and culture it to OD at 18°C ​​and 250rpm 600 Value between 3.0-4.0;

[0032] The preparation steps of SOC medium are as follows: add 20g peptone, 5g yeast extract, 0.5g NaCl, 0.186g KCl into 900mL ultrapure water, stir well to dissolve, adjust the pH to 7.0 with 1M HCl, and use ultrapure water to make up the volume to 1L, after autoclaving, add 5mL of sterilized 2M MgCl 2 , add 20mL filter-sterilized 1M Glucose, mix well, and store at 4°C;

[0033] (3) Transfer the culture to a sterilized 50mL tube, and collect the bacteria by centrifugation at 2500g / 5min at 4°C;

[0034] (4) Discard the supernatant, add the buffer that has been pre-cooled at 4°C, and resuspend manually. Do not shake vigorously in this step;

[0035] The preparation method of th...

Embodiment 3

[0040] (1) Take a small amount of bacterial strains and streak on an LB plate without resistance, and culture overnight at 37°C;

[0041] (2) Pick a single clone and culture it in a 1L Erlenmeyer flask containing 250mL SOC medium, and culture it to OD at 18°C ​​and 250rpm 600 Value between 4.0-5.0;

[0042] (3) The preparation steps of SOC medium are as follows: add 20g peptone, 5g yeast extract, 0.5g NaCl, 0.186g KCl into 900mL ultrapure water, stir well to dissolve, adjust the pH to 7.0 with 1M HCl, and use ultrapure water Dilute to 1L, after high temperature and high pressure sterilization, add 5mL sterilized 2M MgCl 2 , add 20mL filter-sterilized 1M Glucose, mix well, and store at 4°C;

[0043] Transfer the culture to a sterilized 50mL tube, and collect the bacteria by centrifugation at 2500g / 5min at 4°C;

[0044] (4) Discard the supernatant, add the buffer that has been pre-cooled at 4°C, and resuspend manually. Do not shake vigorously in this step;

[0045] The prepa...

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Abstract

The invention discloses a preparation method of competent cells with high transformation efficiency, relates to the technical field of microorganisms and solves the problems of inconvenience in operation, low transformation efficiency and low yield in the prior art. The preparation method comprises steps as follows: (1) strains are scribed on an LB panel and cultured overnight at the temperature of 37 DEG C; (2) an SOC culture medium is inoculated with the strains, and the strains are subjected to shake culture at the temperature of 18 DEG C under the condition of 250 rpm until the OD600 value ranges from 2.0 to 5.0; (3) a strain solution is centrifuged in a sterilized tube with the volume being 50 mL at the temperature of 4 DEG C under the condition of 2,500 g / 5 min, a supernatant is discarded, a buffer solution is added to settled strains, and manual resuspension is performed; (4) a product is centrifuged at the temperature of 4 DEG C under the condition of 2,500 g / 5 min, a supernatant is discarded, a proper quantity of buffer solutions and a proper amount of DMSO are added, the OD600 value is 5.0, and the final concentration of DMSO is 7%; (5) the product is subpackaged in a sterile EP tube, put in liquid nitrogen and frozen quickly, and the competent cells are obtained. The preparation method is simple to operate, the yield is high, and the transformation efficiency can be up to (1*10<8>)-(5*10<9>).

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a method for preparing competent cells with high transformation efficiency. technical background [0002] The process of allowing DNA molecules with genetic information to enter host cells is called "transformation"; host cells that are easily accepted by foreign DNA molecules after physical or chemical treatment are called "competent cells". In prokaryotes, transformation is a relatively common phenomenon. Whether transformation occurs between cells depends on the genetic relationship between the donor bacteria and the recipient bacteria in the evolution process, and on the other hand, whether the recipient bacteria Being in a feeling state has a lot to do with it. The state of competent cells directly affects the efficiency of transformation. In order to enable host cells to replicate or express specific DNA or proteins, obtaining competent cells that are easy to transf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/19
CPCC12N1/20
Inventor 万浩强
Owner XIAMEN LIFEINT TECH CO LTD
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