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Bacteria artificial chromosome plasmid extraction method

An artificial chromosome and plasmid technology, applied in bacteria, introduction of foreign genetic material using vectors, DNA preparation, etc., can solve the problems of insufficient yield, BAC plasmid limitation, and low yield.

Inactive Publication Date: 2005-01-26
HEILONGJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is also because of these characteristics that BAC plasmids are greatly limited when they are extracted by conventional methods.
Generally speaking, with the conventional alkaline lysis method, only 0.1-0.4ug of BAC DNA can be obtained from 5ml of BAC cell culture, and the yield is extremely low. Far from enough
Moreover, bacterial artificial chromosomes are usually composed of tens of thousands of plasmids. Conventional methods need to replace centrifuge tubes more than 3 times when extracting a plasmid sample, which means that if a large number of plasmids are extracted, it will take a lot of labor and material resources

Method used

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Experimental program
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Effect test

specific Embodiment approach 1

[0004] Specific embodiment one: this embodiment includes the conventional steps of adding bacterial culture medium, centrifugation, and adding solution I, solution II, solution III, and isopropanol in a centrifuge tube in chronological order, wherein the formula raw material of solution II used is NaOH, SDS and dd.H 2 O, wherein the molar concentration of NaOH is 0.4M, and the SDS concentration is 0.8%; the formula raw material of solution III is that every 100ml solution III contains 60ml of 5M potassium acetate, 28.5ml of glacial acetic acid, H 2 O11.5ml, the pH value of the final solution III is 4.8, and the selected bacterial culture medium is TB medium.

[0005] The preparation of concrete medicine among the present invention:

[0006] TB medium: To prepare per liter of medium, add 2g of tryptone, 24g of yeast extract, and 4ml of glycerol to 900ml of deionized water. After dissolving, autoclave for 20 minutes, and when the solution is cooled below 60°C, add 100ml of ste...

specific Embodiment approach 2

[0010] Specific embodiment two: the extraction step of this embodiment is: a. add 8~12ml TB culture medium in the 15ml sterilized centrifuge tube, after inoculation, place in 37 ℃ constant temperature shaker and cultivate for 18~22 hours; b. .Centrifuge the resulting product at 3700 rpm for 10 minutes in a Beckman GS-6R centrifuge, and pour off the supernatant after the cells are precipitated; c. Add 2ml of solution I to the centrifuge tube, stir up the cell pellet with a large cotton swab, and vortex Shake until a uniform solution is formed; d. Add 2ml of Solution II to the centrifuge tube and stir slowly with a cotton swab until it is evenly mixed, then place it in an ice bath for 10-15 minutes; e. Add 2ml of Solution III to the centrifuge tube and stir until a uniform solution is formed. solution, and then ice-bathed for 15-20min; f. Centrifuge at 3700rpm for 10min in a Beckman GS-6R centrifuge, scrape off the sediment at the bottom of the centrifuge tube with a cotton swab;...

specific Embodiment approach 3

[0011] Specific embodiment three: present embodiment is the comparative experiment that adopts method of the present invention and existing method to do:

[0012] This experimental protocol is an experimental method for extracting bacterial artificial chromosomes provided by the classic experimental manual of molecular biology "Molecular Cloning Experiment Guide (Third Edition)", author: J. Sambrook.

[0013] The specific process is as follows: 1. Use 10 ml of LB medium containing 12.5 ug / ml chloramphenicol and culture BAC-transformed Escherichia coli, and culture overnight at 37° C. with vigorous shaking. 2. Centrifuge at 4000g for 5min at 4°C to collect bacteria. Carefully discard the culture. 3. Add 5ml of pre-cooled STE to each centrifuge tube, and suspend the cell pellet with a pipette. Centrifuge at 4000g for 5min at 4°C to collect bacteria. Carefully discard the culture. 4. Resuspend the cells in 400ul ice-cold solution I, transfer the cells to a pre-cooled centrifu...

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Abstract

A method for extracting BAC plasmid is provided. In the existing method, the solutionII uses NaOH, SDS and dd.H2O as material, in which NaOH mol concentration is 0.4N, SDS concentration is 0.8%, the solutionIII uses 5M potassium acetate 60ml, glacial acetic acid 28.5ml, H2O 11.5ml as material. The invention changes the solution II and solution III, which promote the BAC DNA extraction efficiency. The adoption of TB culture medium promotes greatly the yield. Only one centrifuging tube is used in the invention, which save the labor and time.

Description

Technical field: [0001] The invention relates to a method for efficiently extracting bacterial artificial chromosome (BAC) plasmid. Background technique: [0002] Bacterial artificial chromosome is a synthetic vector based on Escherichia coli fertility (F), which is characterized by low copy number (1-2 molecules / cell), can amplify very large DNA fragments, and belongs to the low-copy stringent type plasmid. It is also because of these characteristics that BAC plasmids are greatly limited when they are extracted by conventional methods. Generally speaking, with the conventional alkaline lysis method, only 0.1-0.4ug of BAC DNA can be obtained from 5ml of BAC cell culture, and the yield is extremely low. Far from enough. Moreover, bacterial artificial chromosomes are usually composed of tens of thousands of plasmids. Conventional methods need to replace centrifuge tubes more than 3 times when extracting a plasmid sample, which means that if a large number of plasmids are e...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/10C12N15/63C12N15/64
Inventor 王志伟顾雪锋李荣田韩俊郭德栋
Owner HEILONGJIANG UNIV
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