71 results about "Alkaline lysis" patented technology

A scalable alkaline lysis process, including procedures and devices for the isolation of large quantities (grams and kilograms) of plasmid DNA from recombinant E. coli cells. Effective, controllable, and economical operation, and consistent low level of host chromosomal DNA in the final plasmid product. Involves a series of new unit operations and devices for cell resuspension, cell lysis, and neutralization.

The present invention describes isolation of plasmid DNA or nucleic acid material from Escherichia coli (E. coli) or other species of microorganisms, or from other sources. The addition of indicator dyes to the alkaline lysis based purification buffers allows for increased ease of visual monitoring of the mixing and lysis steps of the procedure. The yield and quality of plasmid DNA is improved and the chance of error is reduced. The practice may be applied to other nucleic acid purification procedures.

A scalable alkaline lysis process, including procedures and devices for the isolation of large quantities (grams and kilograms) of plasmid DNA from recombinant E. coli cell. Effective, contgrollable, and economical operation, and consistent low level of host chromosomal DNA in the final plasmid product. Involves a series of new unit operations and devices for cell resuspension, cell lysis, and nuetralization.

A scalable process and device for producing a biomolecule, in particular pharmaceutical grade plasmid DNA. The process includes the steps of alkaline lysis and a neutralization. For separating the lysate and the precipitate, the mixture is allowed to gently flow downward through a clarification reactor that is partially filled, in its lower part, with retention material like glass beads, whereby the precipitate is retained on top of and within the retention. In a preferred embodiment of the lysis step, cell suspension and alkaline lysis solution flow through a lysis reactor that is filled with particulate material like glass beads. The process can be run continuosly and fully automated.

The invention provides an alkaline lysis system for preparing plasmid DNA and a combined system. The alkaline lysis system for preparing plasmid DNA contains a bacterial suspension storage tank, an alkaline lysate storage tank and a hollow ultrafiltration column, wherein the hollow ultrafiltration column contains a first cavity and a second cavity which are divided by an ultrafiltration membrane;the alkaline lysate storage tank is communicated with the first cavity; and the bacterial suspension storage tank is communicated with the second cavity. The alkaline lysis system provided by the invention adopts the hollow ultrafiltration column to mix bacterial suspension and alkaline lysate evenly and avoid the problems that the pH value is too high locally and the shear stress is too large owning to the conventional mixing method such as stirring; and the invention provides the alkaline lysis system for preparing plasmid DNA, and the system has good stability, high efficiency and high repeatability. Meanwhile, the plasmid DNA prepared by the alkaline lysis system has higher supercoiled DNA content which is up to more than 90%.

This invention provides a process for the continuous alkaline lysis of a bacterial suspension in order to harvest pDNA. It further provides for optional additional purification steps, including lysate filtration, anion exchange chromatography, triplex affinity chromatogragphy, and hydrophobic interaction chromatography. These optional purification steps can be combined with the continuous lysis in order to produce a highly purified pDNA product substantially free of gDNA, RNA, protein, endotoxin, and other contaminants.

The invention discloses a kit which is suitable for PCR amplification and used for quickly extracting plant genomes, comprising a lysate, a neutralizer and a precipitation liquid. The kit improves and optimizes the traditional alkaline lysis method used for preparing genome and enlarges the applicable range of the alkaline lysis method used for preparing genome. The kit is widely applied to the extraction on the genome of various plants and can quickly extract the genome used for PCR template directly from plant seeds. The kit has obvious detection effect on the target gene of 600bp, has excellent amplification effects on endogenous gene and exogenous gene and can discover the samples with the components to be measured more than 1%. The extraction time for the whole genome is controlled within 21min, thus greatly shortening the extraction time of the genome.

The invention discloses a simple sequence repeat-polymerase chain reaction (SSR-PCR)-based hybrid rape seed purity identification method, which comprises the following steps of: performing sprouting culture on hybrid rape sample seeds to be detected, performing alkaline lysis on the cultured seedlings, simultaneously performing ultrasonic disruption treatment, and adding an extracting buffer solution to obtain a genome DNA solution; performing PCR amplification on genome DNA by using an SSR primer sequence; performing voltage stabilizing electrophoretic separation on a PCR amplification product in agarose gel; performing imaging and tape reading on the PCR amplification product subjected to electrophoretic separation in a gel imaging system, comparing band characteristics of the sample seeds with those of parent seeds, counting seeds with the band characteristics of male parent and the band characteristics of female parent in the sample seeds, and obtaining the purity of the hybrid rape seeds to be detected according to a variety purity formula. The identification method has the advantages of quickness, simplicity, convenience, high throughput, low detection cost, high detection efficiency, stable and reliable detection results and the like.

A scalable process and device for producing a bio molecule, in particular pharmaceutical grade plasmid DNA is described. The process includes the steps of alkaline lysis, neutralization and clarification and can be further extended. For separating the lysate and the precipitate an improved floatation method is disclosed. This method is based on attachment of CO2 bubbles on the precipitate floe. The CO2 is released from a carbonate salt during or after neutralization (acidification). The method of the invention is preferably carried out in an automated continuous mode applying devices for lysis and neutralization and a novel device for completely continuous clarification (separation of floes and clarified lysate).

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.

This invention provides a process for the continuous alkaline lysis of a bacterial suspension in order to harvest pDNA. It further provides for optional additional purification steps, including lysate filtration, anion exchange chromatography, triplex affinity chromatography, and hydrophobic interaction chromatography. These optional purification steps can be combined with the continuous lysis in order to produce a highly purified pDNA product substantially free of gDNA, RNA, protein, endotoxin, and other contaminants.

The invention relates to a method for preparing polypeptide vaccine agent used in foot-and-mouth disease gene project, wherein said agent is recombined particle that prepared by colonizing pig interferon-alpha gene via molecule biological technique into eucaryon expression carrier; the said agent is recombined particle that prepared by colonizing pig is one of interferon-alpha gene group; the eucaryon expression carrier is any DNA expression carrier with eucaryon starter, protocaryon copier, and protocaryon resistant gene; said inventive particles will transfer into bacillus coli to expand, via alkali crack method to obtain many particles, to be diluted by PBS buffer liquid into some density, to prepare inventive polypeptide vaccine agent. Animal test has proved that said agent is safe coupled with O-tyep vaccine of foot-and-mouth disease gene project, while the protection ration can reach 100% by one injection.

The invention discloses an extraction method of hepatitis B virus deoxyribonucleic acid (DNA) and belongs to the field of nucleic acid extraction in molecular biology. An alkaline lysis technology is adopted according to particular characteristics of blood and viruses; impurities such as proteins, polysaccharides, lipids and the like are not required to be pre-removed; and the virus DNA is directly extracted through lysis buffer 1 and lysis buffer 2. The method has the characteristics of high efficiency, high speed, simplicity and no pollution, and the whole operation process only lasts for 50 minutes. The obtained hepatitis B virus DNA can be applied to molecular biology experiments such as the common polymerase chain reaction (PCR), fluorescence quantitative PCR and the like.

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.

The invention discloses a rapid nosema pernyi template DNA extraction method and application thereof. The rapid nosema pernyi template DNA extraction method specifically comprises the step of releasing, enrichment, separation and purification of DNA through alkaline lysis and magnetic beads. According to the method, operation is simple and rapid, the cost is low, the flux is high, living pupa sampling can be achieved, the antijamming capability is high, the contamination risk is low, the quality of extracted DNA is high, automatic extraction can be achieved, and the method can be widely applied to molecular diagnosis; the detection flux and the operation convenience can be remarkably improved, the false positive rate of detection can be obviously decreased, and the detection sensitivity and accuracy and the detection efficiency are improved; and through an automatic series nucleic acid extraction instrument, a PCR and a preferred result-viewable PCR technique, a visual and automatic nosema pernyi analysis technique which can integrate batched DNA extraction on a porous plate and molecular diagnosis can be achieved. The rapid nosema pernyi template DNA extraction method and the application thereof have important significance for centralized quarantine of antheraea pernyi granulosis and conservation of genetic resources in the antheraea pernyi industry.

The invention discloses a method for quickly extracting plasmodium DNA in a high through-put mode and application of the method. Particularly, the template extracting method comprises the steps that release, enrichment, separation and purification of DNA are conducted through alkaline lysis and magnetic beads. The method is simple and fast in operation, low in cost, high in through-put, low in contamination risk, high in quality of the extracted DNA, and capable of achieving automation and being widely used in molecular detection, the detection through-put and the operation convenience can be remarkably improved, the false positive rate of detection is obviously reduced, and the detection sensitivity and accuracy and the detection efficiency are improved. The visible and automatic sample diagnosis technology that extraction and detection of a mass of DNA on a perforated plate are integrated can be achieved through series connection of an automatic nucleic acid extractor and a PCR instrument and the PCR technology which is visible in optimizing result. The problems that in an existing plasmodium detection technology, the preparation of the target DNA is complex, wastes time, is low in through-put and the like are solved, and the method has a wide application prospect.

The invention discloses a high-flux identification method for purity of Xinjiang thick-peel muskmelon Huangpi 9818 hybrid variety based on an SSR molecular marker. The method comprises the following steps: extracting DNA by adopting an alkaline lysis method, placing seeds in a 96-pore PCR plate, adding a solution buffer A, performing the hot bath for 10 minutes at 95 DEG C in a PCR instrument, then adding a buffer B, uniformly mixing, wherein the obtained solution can be used for PCR amplification. A polymorphism SSR primer is screened by utilizing parents, F1 is used for verification, 8 pairsof parental complementary type primers are screened. The sequencing and combination are performed according to the size of a PCR amplification product fragment, the PCR amplification can be separately performed, the PCR products are mixed, the electrophoresis is performed in a sheet of polyacrylamide gel, after the silver staining, a strip is read, and then the purity of the hybrid variety is identified. The identification result is basically consistent with the traditional purity identification result of the field hybrid variety. By adopting the method, the muskmelon Huangpin 9818 hybrid variety purity can be rapidly and accurately identified by virtue of high flux, the cost is low, and the application prospect is wide.

The invention provides a preparation process for gene vaccines containing UreB and CagA protein of helicobacter pylori. The preparation process includes: establishing recombinant plasmid pIRES-UreB-CagA by recombinant plasmids pUC-UreB and pUC-CagA according to the genetic engineering technology, transforming, screening, amplifying and determining; chemically transforming attenuated Salmonella typhimurium LB500 to perform methylation; screening ampicillin resistant bacterial colony; amplifying selectively; extracting a small quantity of plasmids by the alkaline lysis method; obtaining definitive host bacteria SL7207 by further pulse cell transfection; picking resistant colone to amplify the same in LB culture solution containing 100ug / ml ampicillin to obtain protein; and finally obtaining helicobacter pylori vaccines by purifying the protein. The novel vaccines containing UreB and CagA protein of helicobacter pylori has immunization effects of both UreB and CagA protein, so that immunization efficacy can be improved to a high level.

The subject invention provides a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of mixtures. In a preferred embodiment the procedure is used in forensic DNA applications such as, for example, DNA testing in the case of rape.

This invention provides a process for the continuous alkaline lysis of a bacterial suspension in order to harvest pDNA. It further provides for optional additional purification steps, including lysate filtration, anion exchange chromatography, triplex affinity chromatography, and hydrophobic interaction chromatography. These optional purification steps can be combined with the continuous lysis in order to produce a highly purified pDNA product substantially free of gDNA, RNA, protein, endotoxin, and other contaminants.

A scalable process and device for producing a biomolecule, in particular pharmaceutical grade plasmid DNA. The process includes the steps of alkaline lysis and a neutralization. For separating the lysate and the precipitate, the mixture is allowed to gently flow downward through a clarification reactor that is partially filled, in its lower part, with retention material like glass beads, whereby the precipitate is retained on top of and within the retention. In a preferred embodiment of the lysis step, cell suspension and alkaline lysis solution flow through a lysis reactor that is filled with particulate material like glass beads. The process can be run continuously and fully automated.

The invention discloses a method for preparing a recombinant avian adeno-associated virus. The method comprises 1, preparing three recombinant baculovirus transfer vectors by a DNA recombination technology, 2, respectively carrying out transposition on the three recombinant baculovirus transfer vectors to a DH10Bac competent cell, carrying out screening, and extracting three recombinant shuttle vector plasmid DNAs through an alkaline lysis method, 3, uniformly mixing the three recombinant shuttle vector plasmid DNAs and a transfection reagent PEI according to a certain proportion, transfecting a Sf9 insect cell with the mixture, and after transfection for three days, preparing the recombinant avian adeno-associated virus expressing a human lysozyme. The invention also provides the use of the recombinant avian adeno-associated virus expressing a human lysozyme. The titer of the recombinant avian adeno-associated virus is ten times that of a recombinant avian adeno-associated virus obtained by the conventional method. The human lysozyme expressed by the recombinant avian adeno-associated virus can produce antibacterial effects on the common pathogens.

The invention relates to a reverse transcription method, a reverse transcription kit and application thereof. The reverse transcription kit contains NP-40 lysate or an alkaline lysis agent; the lysis agent is the NP-40 lysate; the formula of the NP-40 lysate comprises RNase-Free H2O, NP-40 and RNasin, wherein the mass concentration of the NP-40 is 0.2-5%, and the concentration of the RNasin is 1-10U; the formula of the alkaline lysis agent is prepared from RNase-Free H2O and KOH, wherein the concentration of the KOH is 0.00001-0.01M; the reverse transcription method comprises the steps of firstly, carrying out lysis on a sample by using the lysis agent; then, directly carrying reverse transcription on the sample, subjected to lysis, by using a reverse transcription reagent. The method is a brand-new method for acquiring complementary deoxyribonucleic acid (cDNA) from the sample; after the method is adopted, the operation process is simplified, the operation difficulty is reduced, the experimental time is greatly shortened, and the cost is lowered.

The invention discloses plasmid DNA alkali cracking equipment. The equipment comprises a bacterial liquid pipeline; a buffer solution pipeline; a bacteria liquid suspension circulating device comprising circulating pipelines which are connected end to end to form a loop, and connected with the bacteria liquid pipeline and the buffer solution pipeline, wherein a pump and a valve arranged on the circulating pipeline; an alkali lysate pre-preparation pipeline; a lysis reactor; a neutralization reactor; an acid liquor pipeline; and a collecting device. The invention also discloses a method for extracting plasmid DNA by using the method. The method comprises the following steps: resuspending thalli by using a bacterial liquid suspension circulating device, pre-preparing an alkali lysate, breaking cells, inputting an acid solution, neutralizing a bacterial liquid and the like. The bacterial liquid can repeatedly circulated in the bacterial liquid suspension circulating device, the flow is improved, and the bacterial liquid resuspension effect is improved; the flow is adjusted, and thus the bacterial liquid can reach the target flow, and is convenient to mix with the alkali lysate.

This invention is fake unicell bacteria Na+ / H+ antiport albumen gene and its clone method, it relates to one kind of gene clone. One kind of fake unicell bacteria Na+ / H+ antiport albumen structure gene and one kind of quickly and economic clone method are provided, gene length is 1089bp, coding 362 amino acids. Procedures are that extra anti-salt fake unicell bacteria is inoculated and cultivated, whole DNA is extracted, primer is designed, PCR reaction and amplified product is gel imaged system sweep record, then the amplified about 1.1kb tripe is cut down. One gland piao lin deoxyribonucleotide (A) is added to end of PCR product, above clear liquid is abandoned, and 20muL weight distilled water is added to dissolve DNA. Carrier is connected with DNA to form single colony. White colony is selected and recombination particle is extracted by caustic cracking solution. Goal gene is identificated and anti-salt level of transformant is detected, clone gene of transformant is sequenced and weight plasmid of transformant is identificated, then colone gene is estimated.

The invention relates to a method for extracting plasmids with endotoxin removed on a large scale. The method comprises the steps of acquiring roughly-separated plasmid DNA by virtue of a classical alkaline lysis method, sequentially adding isopropanol, an NH4Ac solution, RNase A and NaCl, adding a mixed solution of PEG6000 and NaCl, dissolving and precipitating by virtue of a TE solution, repeatedly precipitating by virtue of absolute ethyl alcohol, washing and precipitating by virtue of 70% alcohol, simultaneously coordinating with centrifugation, drying, and finally dissolving and precipitating by virtue of the TE solution, so as to obtain target plasmids. According to the method, the pollution caused by endotoxin to plasmid DNA can be well removed, all reagents are standing chemical reagents in a laboratory, and the operation method is simple, easy to control and suitable for large-scale extraction of plasmids. The concentration of the plasmids can reach 5mg / mL, the endotoxin is lower than 5EU / mu g, and the plasmids can be applied to digestion, DNA sequencing, cell transfection, virus packaging and clinic animal immunization experiments.