413 results about "Lysis buffer" patented technology

The present invention is directed to a method for adsorbing, i.e. non-covalently binding, nucleic acids to a solid phase using a two-step procedure. Furthermore, the present invention pertains to a method for isolating nucleic acids from a biological sample. In the first step of the procedure, lysis is effected by mixing the biological sample with an aequous lysis buffer containing a chaotropic agent and incubating the mixture; in the second step, the concentration of the chaotropic agent in the mixture is increased and the mixture is contacted with the solid phase, whereby the nucleic acids in the liquid phase is adsorbed to the solid phase.

Aiming at the conditions that heparin has severe bleeding side effects in clinical practice and clinical application thereof is restricted, the invention discloses a technique for producing ultra-low molecular heparin sodium (calcium). The technique comprises the following steps of: taking heparin sodium solution, adding sodium nitrite solution for cracking; adjusting the lysis buffer by using alkaline; absorbing impurities by using an anion-exchange column; washing for obtaining ultra-low molecular heparin calcium; carrying out filtration by using an ultrafiltration membrane and obtaining a precipitate by using alcohol; and after desalting, dehydration, re-precipitation, cooling and drying, obtaining a finished product of ultra-low molecular heparin calcium. The product has better and safer antithrombotic effect under low level of anticoagulation, and can be widely used for preventing and treating diseases such as deep vein thrombosis, pulmonary embolism, disseminated intravascular coagulation, and the like.

The invention discloses a nucleic acid extraction and purification method based on nanometer magnetic beads, comprising the following steps: mixing a biological sample and a lysis buffer to make nanometer magnetic beads in the lysis buffer and nucleic acid DNA/RNA which moves into the lysis buffer form a magnetic bead-nucleic acid compound; transferring the compound under the action of a magnetic field to a washing buffer to wash off impurities on the magnetic bead-nucleic acid compound; and transferring the washed magnetic bead- nucleic acid compound under the action of the magnetic field to an elution buffer so as to elute and recover nucleic acid. The nanometer magnetic beads used in the invention have advantages of uniform size, smooth surface, large surface area ratio, high adsorption capacity of nucleic acid, fast magnetic response speed and rapid separation, and can be stored together with the lysis buffer at room temperature for a long time. The extracted nucleic acid DNA/RNA has high purity, is complete and can be directly used for follow-up detection. The method provided by the invention has shorter nucleic acid extraction time than a general magnetic bead method by the use of a nucleic acid extraction reagent, is more suitable for automation and is adopted to realize high-flux nucleic acid DNA/RNA extraction.

The entire process of reverse transcription-polymerase chain reaction (RT-PCR) is simplified by using oligonucleotide-immobilized microplates made of, e.g., polypropylene, to which oligonucleotides are securely immobilized and which can be subjected to thermal cycles of PCR. RT-PCR is preferably conducted in solid-phase. Capturing of mRNA and RT-PCR can be conducted in the same plates. The cDNA synthesized from the mRNA captured on the microplates can be used more than once. Further, in combination with the microplates, a filter plate is used for the preparation of cell lysates wherein target cells are placed on the filter plate, and a lysis buffer is passed through the cell layer on the filter to transfer cell lysate directly to the microplate via well-to-well communication.

The invention provides a protein synthesis system for increasing the expression amount of a foreign protein and an application method of the protein synthesis system. Specifically, a poly-nucleotide sequence or vector constructed by a strong promoter sequence is inserted in front of a nucleotide sequence for encoding an eIF4E binding protein, over-expression of eIF4E binding proteins such as Eap1and p20 can be achieved, and foreign protein synthesis is implemented by using the improved cell lysis buffer. The invention provides the protein synthesis system for increasing the expression amountof the foreign protein, and by adopting the synthesis system provided by the invention, the efficiency of protein translation can be remarkably improved.

The invention discloses a kit and a method for fast separating virus nucleic acid from a biological sample, in order to fast separate virus nucleic acid from a cell-free biological sample. The kit disclosed by the invention comprises a lysis buffer solution, a magnetic bead, a washing buffer solution and an elution buffer solution. The method disclosed by the invention comprises the steps of: disintegrating a biomaterial so that the magnetic bead and the nucleic acid form a complex, separating the complex under the action of an externally-applied magnetic field, removing impurities by washing, and then eluting and recovering the nucleic acid. The method for extracting the nucleic acid is simple, convenient and safe in operation, low in cost, intact in nucleic acid and high in purity, and the nucleic acid can be directly applied to subsequent experiments. The kit can be used for efficiently extracting the virus nucleic acid in combination with an automated nucleic acid extracting instrument and can also be used for extracting the nucleic acid by means of manual operation.

The invention provides an extracting method of viral nucleic acid capable of being used for high flux full automation extraction by a magnetic bead method, which is characterized by comprising the steps of separating viruses via a magnetic bead I: absorbing the viruses to the magnetic bead I from a biological sample to obtain the magnetic bead I with the viruses which is called as a virus-magnetic bead I composite for short, additionally adding a magnetic field to separate the virus-magnetic bead I composite from the sample, suspending the virus-magnetic bead I composite in a lysis buffer I, dissociating the virus from the magnetic bead I in the lysis buffer, additionally adding a magnetic field to remove the magnetic bead I, and heating to crack the virus; and separating a viral nucleic acid via a magnetic bead II: absorbing the viral nucleic acid in a viral lysate via the magnetic bead II, additionally adding a magnetic field to separate the magnetic bead II with the viral nucleic acid from the viral lysate, washing the magnetic bead II with the viral nucleic acid by using a washing solution, and eluting the viral nucleic acid from the magnetic bead II by using a solution to obtain a purified viral nucleic acid. In the invention, cracking cells and separating viruses are completed at one step in one tube by using the magnetic beads; and extraction of the viruses and the viral nucleic acid can be achieved by using two magnetic beads so that the extraction process is simple and rapid without a complicated device, and high pass automatic operation is easy to be realized.

Two pairs of primer are designed: Pl/P3 is an idiocratic primer for pine wood nematode; P2/P3 an idiocratic primer for quasi pine wood nematode. The kit includes 1.25ml solution I,1ml solution II, 100U tag enzyme, and 50ul for each contrapositive DNA of wireworm. The solution I is as following constitutes: Worm Lysis Buffer, 500mM KCl, 100mMTris-ClpH 8.0, 15mM MgCl2, 10mMDTT, 4.5% Tween20, 0.1%gelatin, 0.2ug/ul protease K. The solution II is as following constitutes: 2xPCR reaction Buffer, 4.0mM MgCl2, 0.2mM dNTP, 0.8 mu MP1, P2, P3. The invented kit possesses high specificity, sensitiveness and stability, providing fast, sensitive and specific technical method.

The invention discloses a reagent kit for extracting genome on an automated nucleic acid extraction workstation, and a method thereof, wherein, the reagent kit contains a lysis buffer, a guanidinium buffer, a bead and an eluent; the method comprises the steps of: disintegrating biological tissues or cells by using the lysis buffer, adsorbing DNA by using the bead in the reagent kit, removing impurity by using a cleaning solution and finally recycling the DNA by using the eluent to finish the extraction of the DNA. A regular PCR, a fluorescent quantitation PCR, Genotyping, STR and other various follow-up processing operations can be carried to the extracted DNA. The reagent kit and the method of the invention have the advantages of having wide sample applicability of the reagent, being easy to be operated, and requiring no centrifugalization processing in the whole process, thus being beneficial to the advance of the follow-up operation; the invention can cooperate with the automated nucleic acid extraction workstation to carry out effective DNA extraction and can be used in gene extraction with manual operation.

The invention discloses a novel CTC (circulating tumor cell) enrichment technology and a preparation method of a kit. The technology comprises the steps as follows: through permutation and combination of all possible CD45 isomer protein sequence information, designing antigens, thereby producing multiple antibodies, screening out one antibody capable of recognizing all CD45 isomers, coupling the antibody onto a magnetic bead, and further capturing all leukocytes from blood; collecting 10 ml of peripheral venous blood; removing red blood cells through lysis by an osmosis lysis buffer; mixing a designed specific antibody, the magnetic bead and the rest cell sap containing no red blood cells to separate leukocytes; and obtaining a cell population containing CTCs with higher concentration. According to the technology, a negative collection technology is adopted, so that the proportion of the CTCs is increased substantially, and the rapid and accurate detection is facilitated; meanwhile, a negative screening means is adopted, so that the CTCs are reserved without loss, and the fact that tumor diseases cannot be detected and the treatment for patients is delayed due to the loss of certain CTCs is prevented.

The present invention discloses a kit for detecting aldehyde dehydrogenase 2(ALDH2) gene polymorphism by using the single-tube bidirectional allele-specific amplification technology combined with the SNP sensitivity molecular switch technology, and an amplification method and a detection method thereof. The kit genotypes the Glu487Lys(rs671) site on the aldehyde dehydrogenase 2. The kit includes amplification buffer, polymerase, cell lysis buffer and glycerol, and can complete the genotyping for the SNP site in one PCR reaction so as to understand the genotype of the aldehyde dehydrogenase 2 subject and the product activity thereof.

The invention provides a method of detection of transcription factor expression activity by a luciferase reporter gene system; the method comprises the steps: based on the luciferase characteristics of being lasting, stable and easy to detect, co-transfecting cells with a luciferase reporter gene vector containing a target gene promoter and a transcription factor expression plasmid; after the cells are cultured, carrying out lysis of a certain number of cells with a cell lysis buffer liquid, collecting a lysate containing luciferase, and centrifuging to take a supernatant; and then real-timely detecting luciferase expression intensity data in a luciferase activity detection buffer liquid, at the same time, detecting the luciferase concentration, correcting, and then calculating to obtain the transcription factor expression quantity, namely the transcription factor expression activity needing to detection. The method is simpler and more feasible in whole operation process, shorter in time consuming, higher in flux, relatively high in repeatability and accuracy, and lower in operating costs, thereby having broad application prospects, and having excellent market potential.

The present invention provides a universally applicable lysis buffer comprising a chaotropic 5 agent, a reducing agent, and a proteolytic enzyme suitable for processing a wide variety o different sample types, such as different types of bodily samples relevant for the diagnosis o a respiratory disease. Furthermore, the present invention provides the use of a chaotropic agent, a reducing agent, and a proteolytic enzyme for the lysis of a broad spectrum of bodily samples. Moreover, the present invention provides a method for processing bodily samples which is universally applicable to the lysis of a variety of different types of bodily samples. Furthermore, the present invention provides methods for analyzing a bodily sample or for detecting the presence of a pathogen in a bodily sample, preferably, for diagnosing a respiratory disease, such as pneumonia or tuberculosis. Preferably, these methods are universally applicable to a variety of sample types, are applicable as one-tube-processes, are 15 suitable for performance in a high-throughput setting, and are automatable.

The invention relates to a method for separating free nucleic acid from a blood serum or blood plasma sample by using a magnetic bead, and belongs to the field of nucleic acid purification. The method comprises the following steps: firstly adding the blood serum/blood plasma sample, a lysis buffer, protease K and magnetic bead suspension liquid into a nuclease-free centrifugal tube, fully and uniformly mixing, then performing pyrolysis, adsorbing nucleic acid on the surface of the magnetic bead, performing purification under the effect of an external magnetic field, removing impurities such as protein, polysaccharide, salt and the like through a rinsing step, and finally eluting by adding nuclease-free water to obtain a free nucleic acid solution. The method disclosed by the invention has the characteristics of simplicity, high speed, stability, high efficiency and cheap price; the purified free nucleic acid is complete and high in purity, and can be directly used for subsequent experiments.

The invention relates to an assay for transposase accessible chromatin using sequencing (ATAC-seq) method applied to zebrafish embryos. The method comprises the following steps: firstly, performing zebrafish embryo sample pretreatment; adding lysis buffer into the embryos; cracking the embryos in a water bath by using a wild-neck spearhead; performing a transposition reaction immediately and purifying DNA (Deoxyribose Nucleic Acid); determining a cycle number for library establishment by using a real-time fluorescence quantification method; directly performing library establishment and sequencing by using a further PCR (Polymerase Chain Reaction) method. A regulatory sequence on genome chromatin can be located and decoded by the ATAC-seq method and a DNase-seq (DNase I hypersensitive sites sequencing) method, but the steps in ATAC-seq are simpler, a required cell quantity is smaller, the ATAC-seq is more helpful under the situation that a large quantity of cells cannot be obtained, and the data enriching degree of the ATAC-seq is higher according to an assay result. The ATAC-seq method is further applied to zebrafish embryo cells. Thus, an effective and simple method is provided for the researches of changes of chromatin structures in early developing processes of living organisms and corresponding gene expression regulation and control researches.

The invention discloses an extraction method of total genome DNA from microbes, comprising the following steps of: adding a lysis buffer and lysozyme to a sample to be extracted to enable bacterial cells to fully rupture and release DNA; and extracting the obtained crude extract DNA by phenol/chloroform/isoamyl alcohol extracting solutions to obtain purified total genome DNA of microbes. The extraction method disclosed by the invention has the beneficial effects of big DNA release amount, simple experiment conditions, low cost and the like.

The present invention provides a method of selectively lysing non-viable cells from a cell population within a sample, the method including selectively lysing non-viable cells by mixing a cell-containing sample with a lysis buffer including a non-ionic surfactant and a divalent cation salt.