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Plasmid extraction kit and extraction method

A kit and plasmid technology, applied in the field of molecular biology, can solve the problems of bacterial genomic DNA contamination, organic reagent contamination, low plasmid concentration, etc., and achieve the effects of reliable quality, high recovery rate and good purity

Inactive Publication Date: 2021-02-05
JIANGSU KEYGEN BIOTECH CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional large amount of plasmid extraction kits or reagents will be polluted, one is bacterial genomic DNA pollution; the other is that chloroform-isopropanol needs to be added during the extraction process, and the extracted bacterial plasmids will be polluted by organic reagents
At present, the plasmid extraction kits developed by some international and domestic reagent companies use recovery columns to recover plasmid DNA. However, it is difficult to ensure the quality and purity of plasmid DNA while ensuring the yield of plasmid DNA. As a result, most of the current plasmid extraction reagents The plasmid extracted by the cassette has the disadvantages of low concentration and insufficient purity

Method used

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  • Plasmid extraction kit and extraction method
  • Plasmid extraction kit and extraction method
  • Plasmid extraction kit and extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cultivation of DH5α Escherichia coli carrying pCDNA3.1(+) and pGL3-promoter plasmids respectively.

[0026] Take out the strains or single-clonal plaques from the refrigerator at -80 degrees Celsius, inoculate DH5α Escherichia coli carrying pCDNA3.1(+) and pGL3-promoter plasmids into 1ml medium test tubes respectively, shake and culture at 37 degrees for 8 hours, and rotate 200rpm, after 8 hours, transfer 1ml of cultured bacteria solution into 25ml of culture medium, shake overnight at 37°C (about 15 hours), and rotate at 200rpm. Centrifuge the bacterial liquid cultured overnight at 5000rpm (3800×g) for 5 minutes, discard the medium and keep the precipitate.

Embodiment 2

[0027] Example 2 Plasmid extraction.

[0028] Add 10ml of SI buffer into the cell pellet, and fully suspend the cell. The composition of the S I buffer solution is as follows: 1M Tris-HCl, 0.5M EDTA and 1M glucose, pH=8.0.

[0029] Add 10ml of S II buffer to the suspended bacterial solution, mix slowly (do not shake violently), and lyse. The operation time should not exceed 5 minutes (to prevent genome contamination). The components of the S II buffer are as follows: 2M NaOH and 10% SDS.

[0030] Then add 14ml of S III buffer solution to the lysis system, mix slowly and evenly (do not shake vigorously), and a flake precipitate appears. Centrifuge at 3000 rpm for 10 min, and carefully transfer the supernatant to a new centrifuge tube with a pipette to obtain the supernatant. The composition of SIII buffer is as follows: 3M potassium acetate, 2-3M guanidine hydrochloride and 2M acetic acid.

[0031] Add 18mL of PS cleaning solution to the centrifuge tube, turn over gently se...

Embodiment 3

[0033] Example 3 Plasmid concentration and purity determination.

[0034] Put the adsorption column into a new centrifuge tube and use 0.5ml ddH 2 O (or TE) dissolves the precipitate to obtain a plasmid solution. The A260 is measured by an ultra-micro spectrophotometer, and the quantitative A260 / A280 ratio is generally between 1.8 and 2.0. Plasmid purity was checked by agarose electrophoresis. The TE buffer composition is as follows: 100 mM Tris and 10 mM EDTA (pH 8.0). After testing, the plasmid concentrations of PCDNA3.1(+) and PGL3-promotor extracted by this kit were 700ng / ul and 540ng / ul respectively, which were higher than 560ng / ul and 350ng / ul of the control kit; the values ​​of A260 / A280 were both Between 1.8-20.0.

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Abstract

The invention discloses a high-purity plasmid extraction kit. The high-purity plasmid extraction kit comprises an S I buffer solution, an S II buffer solution, an S III buffer solution, a PS cleaningsolution, an elution buffer solution, a TE buffer solution and a novel centrifugal column, wherein the S I buffer solution comprises Tris-HCl of 1 M, EDTA of 0.5 M and glucose of 1 M, and the pH valueis 8.0; the S II buffer solution comprises NaOH of 2 M and 10% SDS; the buffer solution S III comprises potassium acetate of 3 M, guanidine hydrochloride of 2 M and acetic acid of 2 M, and the pH value is 3.6-4.2; the PS cleaning solution is an isopropyl alcohol solution containing 5-10% of TritonX-114; and the elution buffer solution is 75% ethanol. The invention further provides a method for extracting a large number of plasmids by using the kit. According to the method, the novel centrifugal column is combined with a classic strong base-SDS bacterial cell cracking method, so that a plasmidDNA sample is centrifugally combined to a purification column, plasmid DNA can be fully eluted under a certain condition, rapid purification of plasmids is realized, phenol chloroform extraction is not needed in the whole process, high-purity plasmid DNA can be obtained, and the use of eukaryotic cell transfection can be met.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a plasmid extraction kit and an extraction method. Background technique [0002] Plasmid is an extrachromosomal genetic unit capable of autonomous replication, and bacterial plasmid is one of the most commonly used vectors for recombinant DNA technology. Because of its self-replication ability and the genetic information it carries, it is very practical and valuable for genetic engineering research and its downstream applications in recombinant DNA manipulation. Traditional kits or reagents for a large number of plasmid extractions will be contaminated. One is bacterial genomic DNA contamination; the other is that chloroform-isopropanol needs to be added during the extraction process, and the extracted bacterial plasmids will be contaminated by organic reagents. At present, the plasmid extraction kits developed by some international and domestic reagent companies use recovery co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/10
Inventor 孙益军张峰王雪根李阳韩斌
Owner JIANGSU KEYGEN BIOTECH CORP LTD
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