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DNA molecular weight standard with even 200 bp gradient and rapid preparation method thereof

A technology of molecular weight standards and gradients, applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve problems such as limitations

Inactive Publication Date: 2009-11-11
UNIV OF JINAN
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Problems solved by technology

However, the disadvantages of this method are also very prominent: the size of the DNA fragment produced by enzyme digestion is limited by the type of restriction enzyme used, and the limiting factor is the design level of the artificially constructed plasmid

Method used

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  • DNA molecular weight standard with even 200 bp gradient and rapid preparation method thereof

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Embodiment 1

[0137] Utilize the superplasmid pYE9600 to prepare the preparation method for producing the even-numbered 200bp DNA molecular weight standard gradient, specifically comprising the following steps:

[0138] 1. Take 2-5 microliters of the pYE9600 plasmid stored at -20°C and transfer it into E. coli DH5α or JM109 by heat shock transformation method, spread it on a plate containing ampicillin, and place it upside down in a 37°C incubator Cultivate overnight, pick a single colony the next day, culture with LB liquid medium (5ml) containing ampicillin to OD600=0.8, transfer to 100ml LB liquid medium containing ampicillin, shake vigorously for 3-5 days After 1 hour, when OD600=0.6, continue to transfer to LB liquid medium (1000ml) containing ampicillin that has been preheated at 37°C at a ratio of 1:100 and culture for 12-16 hours.

[0139] 2. A large amount of preparation and purification of the target plasmid pYE9600, the process includes the following steps:

[0140] (1) Harvest ...

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Abstract

The invention relates to a DNA molecular weight standard with an even 200 bp gradient and a rapid preparation method thereof. A super plasmid is constructed by a brand-new genetic engineering method; the DNA molecular weight standard with the even 200 bp gradient is prepared by methods of bacillus coli fermentation and restricted enzyme digestion and is a restricted enzyme digestion mixture comprising seven DNA bands with the lengths of 400 bp, 600 bp, 800 bp, 1000 bp, 14000 bp, 2000 bp and 3000 bp, and the densities (masses) of the seven DNA bands have a certain proportional relation of 2:3:4:5:7:10:15. The rapid preparation method of the DNA molecular weight standard with the even 200 bp gradient comprises the following steps: enabling the seven DNA bands to use a plasmid pYE 9600 as an enzyme digestion object, extracting a target plasmid pYE9600 by methods of bacillus coli fermentation and alkaline lysis, purifying the target plasmid pYE9600 and using a restricted enzyme EcoRI for enzyme digestion so as to release 200 bp serial sections contained in the target plasmid pYE9600.

Description

technical field [0001] The invention relates to the fields of molecular biology reagent preparation and genetic engineering application, specifically constructing a superplasmid by means of genetic engineering, and preparing an even 200bp gradient DNA molecular weight standard by means of Escherichia coli fermentation and restriction endonuclease digestion. Background technique [0002] In the process of DNA synthesis and various methods of genetic manipulation, it is necessary to monitor the size change of the synthesized or manipulated DNA molecule. The commonly used method is to mix the DNA molecule to be tested with a DNA mixture standard of known molecular weight ( Molecular weight standards, DNA markers) are electrophoresed at the same time, and the size of the DNA sample molecule is judged by comparing the migration distance between the DNA molecule to be tested and the DNA molecular standard during the electrophoresis process. Therefore, it is necessary to prepare so...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12P19/34C12N15/10
Inventor 叶春江
Owner UNIV OF JINAN
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