Bacterial lysate and kit for extracting plasmid DNA and method for extracting plasmid DNA

A technology for extracting plasmids and lysates, which is applied in the biological field and can solve problems such as high cost and complex lysate formulations

Active Publication Date: 2019-03-15
河南普诺易生物制品研究院有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the kit and method are suitable for Escherichia coli, the formula of the lysate is complicated and the cost is high

Method used

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  • Bacterial lysate and kit for extracting plasmid DNA and method for extracting plasmid DNA
  • Bacterial lysate and kit for extracting plasmid DNA and method for extracting plasmid DNA
  • Bacterial lysate and kit for extracting plasmid DNA and method for extracting plasmid DNA

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Embodiment 2

[0043] The cell lysate for extracting plasmid DNA in this example consists of lysis buffer, lysozyme, and RNase A. Each 100 mL of cell lysate includes 250 mg of lysozyme and 15 mg of RNase A. Each 100 mL of lysis buffer contains 1.5 g of sucrose. , 0.7g disodium edetate, 0.3g tris(hydroxymethyl)aminomethane hydrochloride, 2.7gNH 4 Cl, 0.1 mL 0.5% Triton X-100, 0.2 g CaCl 2 , 0.5g guanidine hydrochloride, 0.5g guanidine isothiocyanate, and the balance is water, and the pH of the cell lysate is 8.0.

Embodiment 3

[0045] The bacterial cell lysate that this embodiment extracts plasmid DNA is made up of lysis buffer, lysozyme, RNase A, includes lysozyme 300mg, RNase A 20mg in every 100mL bacterial cell lysate, contains 5g sucrose, RNase A in every 100mL lysis buffer, 1g disodium edetate, 0.4g tris(hydroxymethyl)aminomethane hydrochloride, 4.2g NH 4 Cl, 1.5 mL 0.5% TritonX-100, 0.31 CaCl 2 , 2.3g guanidine hydrochloride, 1.4g guanidine isothiocyanate, and the balance is water, and the pH of the cell lysate is 6.6.

Embodiment 1

[0047] The kit for extracting plasmid DNA in this embodiment includes bacterium lysate, rinsing solution I, rinsing solution II and eluent, and the bacterium lysate in this embodiment is the bacterium lysate for extracting plasmid DNA in Example 1 bacterial cell lysate. Wherein, the composition of rinse liquid I is: every 100mL rinse liquid I contains 76.4g guanidine hydrochloride, 0.8g tris(hydroxymethyl)aminomethane hydrochloride, 70mL absolute ethanol, and the remainder is water. The pH at 25°C is 7.0; the composition of rinse solution II is: every 100mL rinse solution II contains 0.15g NaCl, 0.08g tris(hydroxymethyl)aminomethane hydrochloride, 0.4g disodium edetate , 60mL of absolute ethanol, and the balance is water, and the pH of the rinse solution II at 25°C is 8.0; the eluent consists of: 0.16g tris(hydroxymethyl)aminomethane in every 100mL eluent Hydrochloride, 0.04g disodium ethylenediaminetetraacetic acid, the balance is water, and the pH of the eluent at 25°C is 8...

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Abstract

The invention relates to a bacterial lysate and a kit for extracting plasmid DNA and a method for extracting the plasmid DNA, and belongs to the technical field of biology. The bacterial lysate for extracting the plasmid DNA comprises lysis buffer, lysozyme and ribonuclease A(RNaseA); wherein100 mL of the lysis buffer comprises 1.5-10 g of sucrose, 0.7-2.2 g of ethylene diamine tetraacetic acid (EDTA), 0.3-0.9 g of tri(hydroxymethyl) amino methane hydrochloride, 2.7-5.4 g of NH4Cl, 0.1-2.0 mL of 5% Triton X-100, 0.2-0.6 g of CaCl2, 0.5-8 g of guanidine hydrochloride, 0.5-3 g of guanidinium isothiocyanate and the balance of water. The kit for extracting the plasmid DNA comprises the bacterial lysate, rinsing solution and eluant and is low in cost, high in purity of extracted plasmid and high in applicability.

Description

technical field [0001] The invention relates to a cell lysate for extracting plasmid DNA, a kit and a method for extracting plasmid DNA, belonging to the field of biotechnology. Background technique [0002] Plasmid DNA is a commonly used gene delivery tool in molecular biology experiments, and is widely used in the field of genetic engineering. The separation and extraction of plasmid DNA is a routine technique in molecular biology experiments. Its extraction efficiency, purity and quality directly affect later molecular biology experiments, such as enzyme digestion, ligation, transformation of Escherichia coli, and transfection of mammals. The success or failure of processes such as cells, PCR amplification, and sequencing. [0003] The method for extracting plasmid DNA from prokaryotes includes three basic steps: culturing bacteria to amplify the plasmid; collecting and lysing the cells; and isolating and purifying the plasmid DNA. There are three commonly used methods ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 郭潇王天云窦媛媛杨献军王建华
Owner 河南普诺易生物制品研究院有限公司
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