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Rapid covalently closed circular DNA purification kit

A kit and circular technology, applied in the field of rapid covalently closed circular DNA purification kits, can solve the problems of not being able to effectively prevent plasmid DNA damage, not being suitable for EndA wild-type strains, and not recommending purification of plasmid DNA, etc., to reduce Possibility of contaminating other disease sources, fewer steps, and shorter operating time

Inactive Publication Date: 2011-02-23
徐堤
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AI Technical Summary

Problems solved by technology

[0011] Its disadvantages are: (1) It is not suitable for Escherichia coli strains that can release a large amount of carbohydrates after denaturant, lysozyme and heat treatment
(2) For E. coli strains expressing DNase, it is not recommended to purify plasmid DNA by boiling
(3) During large-scale extraction, the bacterial lysate is too viscous and difficult to handle. When cultivating bacteria, chloramphenicol must be added to the culture medium
The disadvantage of this method is that it is not applicable to all strains
[0014] Compared with the one-step plasmid DNA purification technology of the present invention, the method has a very similar operation process. The biggest difference is that the Eppendorf method needs to use animal-derived lysozyme to lyse bacteria, and animal-derived RNase to remove RNA. Due to the above-mentioned price and the possibility of introducing pathogens and other factors, this method is obviously difficult to use in industrial-scale large-scale plasmid DNA purification
Secondly, the Eppendorf method uses a mild lysis method based on lysozyme, and the effect of lysing bacteria is definitely not as good as the chemical lysis solution containing guanidine isothiocyanate and SDS used in the present invention, and the recovery rate of plasmid DNA will not be very high.
In addition, the mild lysate has weak inhibition on DNase and cannot effectively prevent its damage to plasmid DNA, so it is not suitable for EndA wild-type strains

Method used

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  • Rapid covalently closed circular DNA purification kit

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1: A fast covalently closed circular DNA purification kit is characterized in that: it is completed by the following steps and methods, and the specific steps and methods are as follows,

[0061] The steps of covalently closed circular DNA purification technology are as follows:

[0062] Step 1: Bacterial precipitation. Since the volume of bacteria is 10-50L, the bacterial precipitation is mainly collected by filtration, and the liquid medium is removed to obtain bacteria;

[0063] The second step: non-alkali lysis, using non-alkali lysis solution to lyse the bacteria, during which a large stirrer is required to fully stir to lyse the bacteria, and at the same time, the polyamines make the genomic DNA and RNA highly concentrated and lose the ability to combine with the silica gel membrane. We have optimized the formula of non-alkali lysate, adding spermine and hexammine cobalt;

[0064] Step 3: The silica gel membrane is combined with circular DNA, and the l...

Embodiment 2

[0176] Example 2: Extraction of animal mitochondrial DNA in 2 steps:

[0177] Animal mitochondrial DNA extraction (the first step is total DNA extraction)

[0178] (1) Cell lysis buffer:

[0179] Tris(pH8.0)100mmol / L

[0180] EDTA(pH8.0)500mmol / L

[0181] NaCl 20mmol / L

[0182] SDS 10%

[0183] Trypsin 20ug / ml

[0184] (2) Proteinase K: Weigh 20mg proteinase K and dissolve it in 1ml sterilized double distilled water, set aside at -20°C

[0185] (3) TE buffer (pH8.0): autoclave, store at room temperature

[0186] (4) Phenol: chloroform: isoamyl alcohol (25:24:1)

[0187] (5) Isoamyl alcohol, cold absolute ethanol, 70% ethanol, sterilized water

[0188] (1) Take 0.1g (0.5cm) of fresh or frozen animal tissue 3 ), cut as much as possible. Place in a glass homogenizer, add 1ml of cell lysis buffer to homogenize until no tissue pieces are seen, transfer to a 1.5ml centrifuge tube, add 20ul of proteinase K (500ug / ml), and mix well. Bathe in a constant temperature water bat...

Embodiment 3

[0204] Example 3: Plant chloroplast mitochondrial DNA extraction is divided into two steps, the first step is to extract the total plant DNA, and the second step is to extract mitochondrial chloroplast DNA from the total DNA.

[0205] Plant mitochondrial chloroplast DNA extraction (first step)

[0206] SDS Modified DNA Extraction Solution

[0207] (1) Solution Ⅰ: NaCl (100mmol / L), Tris-HCl (50mmol / LpH 8.0), EDTA (50mmol / LpH8.0).

[0208] (2) Solution Ⅱ: NaCl (100mmol / L), Tris-HCl (50mmol / LpH 8.0), EDTA (50mmol / LpH8.0), SDS (1%)

[0209] (3) PCA: Mix Tris saturated phenol with chloroform and isoamyl alcohol at a volume ratio of 25:24:1, place in a brown reagent bottle, and store at 4°C.

[0210] (4) CA: Mix chloroform and isoamyl alcohol at a volume ratio of 24:1, place in a brown reagent bottle, and store at 4°C.

[0211] (5) 5M NaCl

[0212] (6) Absolute ethanol

[0213] (7) 1×TE buffer solution (pH 8.0): 1 mol / LTris-HCl (pH 8.0) 1ml, add 0.5 mol / LEDTA (pH 8.0) 0.2ml, di...

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Abstract

The invention relates to a rapid covalently closed circular DNA purification kit. The kit is finished by a method which comprises the following steps of: bacterial precipitation, non-alkaline cracking, combining circular DNA by a silicone membrane, column elution, and elution; on the basis of the method, components such as hexamminecobalt chloride and spermine are added; bacteria are cracked by adopting improved non-alkaline cracking liquid; linear genome DNA and RNA are highly concentrated by using polyamine to lose the capability of combining the silicone membrane selectively, so that the genome DNA and RNA can still combine the silicone membrane; and a rapid covalently closed circular DNA purification kit can be achieved by washing and eluting. The DNA purification technology of the invention replaces two-step DNA purification technology, completely saves a plasmid crude extraction step, does not depend on the insensitivity of the species of escherichia coli to the acuteness degree of an operation, has less steps, shortens the time of the purification operation of plasmid DNA, saves manpower and material resources, and is suitable to be used in high-flux plasmid DNA purification and industrial large-scale plasmid DNA purification technology.

Description

Technical field: [0001] The invention relates to a fast covalently closed circular DNA purification kit. Background technique: [0002] Plasmid DNA is used in molecular biology research almost every day, and it also needs to be purified frequently. In addition, in the field of gene therapy and gene vaccines, as more and more gene therapy drugs and gene vaccines enter clinical trials and industries In the stage of culture, the demand for plasmid DNA is also increasing. Because the quality standard of plasmid DNA for clinical use is different from that of plasmid DNA for molecular biology research, and the purification process of small-scale laboratory and large-scale industrial purification process are also different, so a good plasmid DNA purification technology must meet the differences in various fields. need. At present, only the plasmid DNA purification method based on alkaline lysis can meet this requirement. Another method, the boiling method, is mainly used in the f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 徐堤
Owner 徐堤
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