Binding liquid and superhelix plasmid large extraction kit based on binding liquid

A kit and supercoiled technology, applied in the field of supercoiled plasmid extraction kits, can solve the problems of potential safety hazards, less supercoiled plasmids, pollution, etc., and achieve improved recovery and purity, enhanced binding efficiency, and simple operation Effect

Pending Publication Date: 2022-05-17
合肥欧创基因生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the large plasmid extraction kits or reagents developed by some reagent companies at home and abroad have pollution problems: first, the extracted plasmid bacterial genomic DNA is contaminated, which will have adverse effects on downstream experiments; second, it needs to be added during the extraction process. Toxic reagents such as chloroform-isopropanol have potential safety hazards; the third is that there are few supercoiled plasmids extracted

Method used

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  • Binding liquid and superhelix plasmid large extraction kit based on binding liquid
  • Binding liquid and superhelix plasmid large extraction kit based on binding liquid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The invention provides a binding solution for plasmid extraction, which can enhance the binding efficiency of the plasmid and the spin column, and increase the content of the extracted supercoiled plasmid. The binding solution contains 1-5M guanidine isothiocyanate, 10-30% (V / V) polyethylene glycol 8000, 60-90mM Tris-HCl, 20-40mM EDTA, 0.5-2M NaCl, and the pH is 4-7; preferably , containing 4M guanidine isothiocyanate, 20% (V / V) polyethylene glycol 8000, 75mM Tris-HCl, 32mM EDTA, 1M NaCl, pH 5.9.

[0037] The positive impact of the binding solution provided in this example on plasmid extraction will be demonstrated through comparative experiments below.

[0038] 1. Take the LB liquid medium (overnight culture) containing the target plasmid, centrifuge at 8000rpm for 5min, collect 300ml of bacterial liquid precipitate, discard the supernatant, then invert the centrifuge tube on absorbent paper, and remove the residual supernatant;

[0039] 2. Divide 300ml of bacterial l...

Embodiment 2

[0052] The invention provides a supercoiled plasmid large-scale extraction kit, which is superior to commercially available kits in terms of extraction volume and recovery rate, and will be verified by comparative experiments below.

[0053] 1. Take the LB liquid medium (overnight culture) containing the target plasmid, centrifuge at 8000rpm for 5min, collect 600ml of bacterial liquid precipitate, discard the supernatant, then invert the centrifuge tube on absorbent paper, and remove the residual supernatant;

[0054] 2. Divide 600ml of bacterial liquid precipitation into twelve centrifuge tubes B1-B6 and C1-C6 on average, use the present invention to extract samples B1-B6, and use commercially available plasmid extraction kits from well-known Q companies to sample C1 -C6 for extraction;

[0055] 3. Add 7ml of resuspension solution to each of samples B1-B6, shake and mix until the resuspension solution precipitates and there are no small bacterial lumps;

[0056] 4. Add 600 μ...

Embodiment 3

[0073] In this example, the stability test of the supercoiled plasmid large-scale extraction kit provided by the present invention is carried out.

[0074] 1. Take the LB liquid medium (overnight culture) containing the target plasmid, centrifuge at 8000rpm for 5min, collect 400ml of bacterial liquid precipitate, discard the supernatant, then invert the centrifuge tube on absorbent paper, and remove the residual supernatant;

[0075] 2. Divide 400ml of bacterial liquid sediment into eight centrifuge tubes D1-D8;

[0076] 3. Add 7ml of resuspension solution to samples D1-D8, shake and mix until the resuspension solution precipitates and there are no small bacterial lumps;

[0077] 4. Add 7ml of lysate and gently invert it up and down 6-8 times until the solution is uniform blue;

[0078] 5. Add 7ml of neutralizing solution and gently invert it up and down 6-8 times until the solution turns from blue to yellow and flocculent precipitation appears;

[0079] 6. Centrifuge at 10,...

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Abstract

The invention discloses a binding solution and a superhelix plasmid large extraction kit based on the binding solution. The binding solution comprises 1-5 M of guanidine isothiocyanate, 10-30% (V / V) polyethylene glycol 8000, 60-90 mM of Tris-HCl, 20-40 mM of EDTA and 0.5-2 M of NaCl, and the PH is 4-7. According to the superhelical plasmid large extraction kit based on the binding liquid, firstly, escherichia coli containing target plasmids is subjected to resuspension, splitting decomposition and neutralization through an alkali splitting decomposition method; adding a binding solution, uniformly mixing, and standing at room temperature to form a mixed solution; centrifuging, filtering the mixed solution through a centrifugal column, and discarding the filtrate; and finally, washing and eluting the plasmids adsorbed on the centrifugal column to obtain the target plasmids. The special binding liquid can enhance the binding efficiency of the plasmids and the centrifugal column and improve the content of the extracted superhelical plasmids.

Description

technical field [0001] The invention relates to the technical field of plasmid extraction, in particular to a supercoiled plasmid large-scale extraction kit. Background technique [0002] Plasmid is a genetic unit capable of autonomous replication outside the chromosome, and bacterial plasmid is one of the most commonly used vectors for recombinant DNA technology. The self-replicating ability of plasmids and the genetic information they carry make them very practical and valuable in genetic engineering research and its downstream applications. Plasmid extraction is one of the most critical steps in genetic engineering technology, which is related to the smooth progress of subsequent transformation, DNA sequencing, PCR, enzyme digestion and other experiments. [0003] At present, the improved alkaline lysis method is the most commonly used and most effective plasmid extraction method. The basic principle is that when the bacteria are lysed in NaOH and SDS, the protein and DN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/101C12Q1/6806C12Q2527/125C12Q2523/308C12Q2523/32Y02A50/30
Inventor 卢德景夏梦凡王美玲周艳雷杨芳梅修朝阳高强郑宝富
Owner 合肥欧创基因生物科技有限公司
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