69 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 from bacteria. The addition of dyes to the alkaline lysis based purification buffers (P1, P2, and P3) allows for improved visual monitoring of the steps of preparing a bacterial lysate filtrate coupled to filtration or spin-column chromatography. The method comprises the suspending of the bacterial cells with buffer P1 (suspension is red / pink); lysing the bacteria with buffer P2 (suspension goes from red / purple color to translucent / purple); precipitating cellular debris with buffer P3 (solution becomes yellow with debris suspension); centrifuging or filtering to product a lysate filtrate; binding the lysate filtrate to a DNA binding matrix; washing; and isolating the plasmid via chromatography. The yield and quality of plasmid DNA is improved due to more consistent lysis. Errors in buffer addition are reduced by visualizing the color as buffers are added and also of changes in color of the preparation at each step.

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.

Apparatus, reagents, and methods for isolating plasmid DNA from bacteria by alkaline lysis using a solid or immobilized P2 and / or P3 reagent in combination with a DNA-binding matrix.

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 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 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 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 andoptimizes 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.