403 results about "Protease K" patented technology

The invention discloses a reagent for extracting and purifying DNA, comprising the following substances: preprocessing lysis solution with pH of 7.4-8.5: water serves as solvent, and solute comprises the substances with the following final concentration: 10-100mM of buffer salt, 0.5%-5% of surface active agent, 1-100mM of chelating agent, 50-150mM of soluble salt, and 0.2-4mg/ml of protease K; pyrolysis combined liquid with pH of 6.4-7.4: water serves as solvent, and solute comprises the substances with the following final concentration: 20-150mM of buffer salt, 3-8 of MChaotropic salt, 1-10% of surface active agent, 0.5-4% of amphoteric ion detergent, 10-100mM of chelating agent and 15-30% of alcohol; cleaning solution I: water serves as solvent, and solute comprises the substances with the following final concentration: 4-6 of MChaotropic salt, 25-50% of alcohol and 10-100mM of chelating agent; cleaning solution II is 75% of ethanol water solution; spent regenerant is TE (10mM Tris-HCl, 1mMEDTA, and pH is 8.0); magnetic bead suspension is nanoscale silicone coated magnetic microsphere (50-100mg/ml).The DNA extracting efficiency is as high as 93.67% by adopting the reagent.

The invention provides a magnetic bead process nucleic acid extraction and transformation kit, and a using method thereof. The kit includes a lysate, a protease K, a magnetic bead solution, a transforamtion reagent, a nucleic acid protection agent, a binding solution, a washing solution I, a washing solution II and an eluent. The using method of the kit includes the following steps: (1) lysis; (2)transformation; (3) layering; (4) binding; (5) primary washing; (6) secondary washing; (7) drying; and 8) elution. The kit and the using method thereby can complete the transformation while achievingautomatic extraction of nucleic acid from a sample. The sample is lysed, releases the nucleic acid, and then begins to transform, the nucleic acid is separated from proteins by the binding solution after the transformation is completed, the methylated nucleic acid is purified by a magnetic bead process, and a pre-denaturation step in subsequent qPCR detection of the sample is used to replace a previously omitted sulfo group removal step, so a purification process in the original nucleic acid extraction step is omitted in the entire extraction and transformation process.

The invention discloses a kit for methylation of colorectal cancer related gene multi-sites and application of the kit. A primer combination and a probe can specifically detect the methylation degrees of a plurality of colorectal cancer related gene sites. The invention further provides application and a method of the primer combination and the probe for detecting the methylation in colorectal cancer and precancerous polyps screening. The method comprises the steps that a cell lysis solution is added to cell sediment and mixed uniformly, then PVPP is added for crude extraction, a cell sediment sample after primary treatment contains a little protein and RNA, protease K and RNA enzyme are added for completely removing the protein and the RNA, cell lysate is added to a centrifugal column with an adsorption film, after impurities are washed by a DNA rising solution, a DNA eluent is added to elute the DNA in the absorption film, and finally a DNA solution is collected through centrifugation. Efficient pipe collection detection is performed aiming at the plurality of gene sites, the flexibility and the specificity are improved, and the tumor early screening requirement can be improved.

The invention relates to a composite for preservation of human saliva and a preparation method of the composite. The composite comprises the following components: Tris-HCl with pH being 6-8.5 (5-20mmol/L), EDTA (Ethylene Diamine Tetraacetic Acid) (0.5-2mmol/L), NaOAc (2.5-3.5mol/L), cane sugar (0.1-0.4mol/L), N-acetyl-5-methoxytryptamine (1-3mmol/L), propylparaben (1-4mg/mL), diazonium imidazolidinyl urea (1-3mg/mL), protease K(10mu g/mL) and a system with pH being 7.5-8.5. When used for preservation of human saliva, the composite is capable of inhibiting nuclease activity and preventing nuclease from being contaminated by external microbes or being oxidized by itself, thereby guaranteeing the integrality and the purity of genomic DNA (deoxyribonucleic acid). The composite is long in preservation period, wide in preservation temperature range and applicable to gene detection and related scientific researches.

The invention discloses a kit for extracting DNA/RNA of a virus by utilizing magnetic beads and a using method. The kit comprises the following eight components: an extracting solution I (cracking), an extracting solution II (binding), an extracting solution III (scrubbing), an extracting solution IV (scrubbing), an extracting solution V (eluting), magnetic bead suspension, a protease K working solution and a nucleic acid settling agent. The kit extraction method comprises the following steps: cracking, binding, scrubbing and eluting. The kit and the extraction method can be used for extracting the DNA/RNA of the viruses of different types of samples, the impurities such as proteins and lipids in the samples are effectively removed, the extracted nucleic acid is high in purity and complete in fragments, and the extraction process is safe and non-toxic.

The invention discloses an FAdV-4 PCR detection kit and detection method. The kit comprises protease K, a lysing solution, sodium acetate, saturated phenol, chloroform, isoamylol, anhydrous ethanol and sterile distilled water, and is characterized by comprising 2*TaqPCR Mix, a sense primer and a reverse primer, wherein the sequence of the sense primer is shown as SEQ ID No.1, and the sequence of the reverse primer is shown as SEQ ID No.2. The kit can be used for detecting FAdV-4. The method for detecting FAdV-4 by adopting the kit is also provided, and FAdV-4 can be detected quickly and specifically.

The invention discloses a nucleic acid extraction method by virtue of a paramagnetic particle method. The nucleic acid extraction method comprises the following steps: sub-packaging 'N' [mu]M (M refers to mol/L) of sample ('N' is less than or equal to 300 and greater than or equal to 50), 'N' [mu]M of lysate and '0.1N' [mu]M of protease K in a No.1 nucleic acid extraction plate, conducting a cracking reaction at 70 DEG C for 10-60min, and then adding '1.1N' [mu]M of magnetic particle-binding buffer; sub-packaging '2.5N' [mu]M of washing solution A, '2.5N' [mu]M of washing solution B and '0.25-0.5N' [mu]M of TE solution in No.2, No.3 and No.4 nucleic acid extraction plates; and under the action of magnetic force, adsorbing magnetic particles in the No.1 nucleic acid extraction plate by virtue of a magnetic bar, conducting transferring, releasing, rapid mixing and magnetic absorption and sequentially processing the magnetic particles by virtue of the No.2, No.3 and No.4 nucleic acid extraction plates, so as to obtain extracted nucleic acid. With the application of the method disclosed by the invention, the influence of impurities on nucleic acid extraction from saliva and other biological samples is effectively reduced, so that nucleic acid extraction efficiency and nucleic acid purity are improved.

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 human feces DNA extraction method. The prior art is complicated and time-consuming; and the trace DNAs extracted by the previous sampling method is too little to perform molecular diagnosis. The extraction method uses selective sample collection PSC equipment for feces samples and adopts a low temperature preservation method. The extraction method comprises the following steps: picking a sample by using a sterilized gun head or sterilized blade and directly placing the picked sample on ice; and adding a feces lysis solution, fully cracking and evenly mixing, adding bovine serum albumin, centrifuging to remove the supernatant, adding protease K to perform a digestion reaction at 58 DEG C, then adding a protein precipitation solution to precipitate the protein, centrifuging to obtain the supernatant, adding a silica gel membrane binding solution to fully bind DNAs, centrifuging to obtain precipitates, adding a silica gel membrane washing solution to wash twice, centrifuging, and adding a TE (Tris + EDTA) buffer solution to finally obtain a preservation solution with DNAs. The human feces DNA extraction method has the advantages of high repeatability and stability, simpleness and short time.

The application discloses a nucleic acid fast extraction kit, which includes a mixed enzyme working solution, a cracking adsorption liquid, a detergent liquid, a rinsing liquid and an eluate liquid that are individually prepared. The mixed enzyme working solution includes protease K and lysozyme. The cracking adsorption liquid includes Gu-HCl, Tris-HCl, EDTA, isopropanol, SDS, Triton-100, beta-mercaptoethanol and sodium citrate. The detergent liquid includes LiCl, NaCl, MOPS and ethanol. The rinsing liquid is ethanol. The eluate liquid is EDTA and Tris-HCl. The kit is suitable for extraction of genomic DNA of whole blood, culture cell, tissues, saliva, G<-> bacteria and G<+> bacteria. The purity of extracted DNA is high, and the DNA is suitable for downstream digestion and PCR biological operation. The kit is stale in reagent property, can meet common laboratory demands and can overcome defects of toxicity, long time consumption and tedious operation of a traditional method.

The invention provides a rapid no-heating nucleic acid extraction kit based on a magnetic bead method. The kit comprises a solution containing magnetic beads, protease K, a polyA solution, deproteinizing liquid, washing liquid, eluant and lysate. The invention also provides a method for extracting nucleic acid by using the kit. The method includes the steps that the lysate and the magnetic beads are added to a sample and fully shaken for uniform mixing, and when lytic cells release nucleic acid, the nucleic acid is captured with the magnetic beads; then, the specific deproteinizing liquid andwashing liquid are adopted for washing respectively to remove protein, salt ions and other impurities on the surfaces of the magnetic beads; finally, the specific eluant is used for rapidly eluting the nucleic acid at room temperature. Compared with a conventional nucleic acid extraction kit based on a magnetic bead method, the kit has the advantages that the steps are simple and rapid, and the whole process only takes about 15 min; besides, heating is not needed, so that the probability of sample room contamination possibly caused by aerosol is reduced; no chloroform phenol or other toxic reagents are used, so that the kit is safe and free of pollution.

The invention discloses a whole blood genomic DNA high-flux plate type extracting kit and an extracting method, and relates to the technical field of biology. The whole blood genomic DNA high-flux plate type extracting kit comprises a lysate 1, a lysate 2, a combination liquid, and magnetic beads, wherein the lysate 1 comprises the following components: 2 to 8 mol/L guanidine hydrochloride, 1 to 10 percent of triton-100 (V/V), 1 to 10 percent of Tween-20 (V/V), 1 to 10 percent of chelex-100 (m/V), 2 to 10 mmol/L ethylene diamine tetraacetic acid and 20 to 100 mmol/L tromethamine, wherein the pH is regulated to be 4.5 by hydrochloric acid, and the solvent is deionized water; the lysate 2 comprises the following components: 20 mg/ml protease K and 50 mmol/L trimethyl aminomethane, wherein the pH is regulated to be 8.0 by the hydrochloric acid; the combination liquid comprises the following components: 1 to 3 mol/L sodium chloride and 40 to 90 percent of isopropyl alcohol (V/V), wherein the solvent is the deionized water. According to the whole blood genomic DNA high-flux plate type extracting kit and the extracting method, the purpose of extracting 96 samples simultaneously within 50 minutes can be achieved without centrifuging or matching an automatic instrument.

The invention relates to a gene mutation type recombined protease K and an industrialized production method thereof. Specifically, the invention relates to the gene mutation type recombined protease K which is obtained by performing gene reformation and protein engineering and has an enzymatic activity being more than two times of the enzymatic activity of the same natural protein, and further relates to the industrialized production method and a technical process for utilizing yeast cells to large-scale culture expressing foreign proteins and prepare the gene mutation type recombined protease K.

The invention discloses a kit and a method for quickly extracting DNA (deoxyribonucleic acid) from colla corii asini. A combined technology of an SDS (sodium dodecyl sulfate)-protease K and Chelex-100 combined process DNA extraction technology and a Glassmilk DNA purification technology is applied to DNA extraction of the colla corii asini. The dosage of needed colla corii asini paste is controlled to be 0.3ml to 0.5ml, the weight of colla corii asini blocks can be controlled to be 0.2mg to 0.6mg, and extracted DNA is high in concentration, excellent in purity, stable and difficult to degrade; due to extracted DNA, sequence fragments of 100bp-500bp can be amplified, and subsequent molecular biology experiments of gene cloning, sequencing and the like can be carried out. According to the method, the operation is simple, the dosage is low, and the consumed time is short; the method is especially suitable for DNA extraction of various types of colla corii asini products with low nucleic acid content and high degradation degree caused by deep processing; a foundation is provided for realizing colla corii asini source identification by adopting a DNA molecular biology identification technology.

The invention relates to a method for quickly and efficiently extracting whole blood genome DNA (deoxyribonucleic acid), and belongs to the field of nucleic acid purification. The method comprises the following steps: adding a red cell lysis solution into whole blood, eddying until red cells are completely split, removing supernatant, and sucking all residual liquid; adding a white cell lysis solution and protease K, and splitting white cells in a water bath of 37 DEG C until the white cells are deposited and dissolved; adding sodium chloride and chloroform, carrying out centrifugal separation on DNA, and transferring the supernatant to another clean EP (epoxy) pipe; adding ammonium acetate and absolute ethanol for depositing DNA, washing by 75% of ethanol, naturally drying, and adding TE (Tris-EDTA(ethylene diamine tetraacetic acid)) for dissolving to obtain DNA. The method has the characteristics of high speed, high efficiency and non-toxicity. The extracted DNA can be used for various subsequent molecular biology studies such as polymerase chain reaction (PCR), methylation detection and gene cloning.

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 discloses an extraction process of black corn silk polysaccharide, which comprises a raw refining process, a protein removing process, a decolorization process and a grading and purifying process, wherein in the raw refining process, protease K water digestion and ultrasound extraction combined method is adopted in the raw refining process, the protein removing process is that sevage method and TDA (trichloroacetic acid) method are alternatively used for deproteinization, an ethanol decolorizing and peroxide decolorizing combined method is adopted in the decolorization process, and DEAE- cellulose (chlorine type) column chromatography method is adopted in the grading and purifying process. Compared with the prior art, the invention has the characteristics of high recovery ratio, excellent decolorization effect, high purity of polysaccharide and the like. The prepared black corn silk polysaccharide has better anticancer and hypolipemic effects.

The invention provides a simple method for efficiently extracting DNA (deoxyribonucleic acid) from soil samples. According to the simple method, the DNA in environmental samples is fully released and exposed by using combination of methods such as glass bead grinding, liquid nitrogen repetitive freeze-thawing, SDS (sodium-dodecyl sulphate), lysozyme and a protease-K method for the different soil samples, the DNA purification is carried out through multiple phenol imitation extraction processes, and the determination of the DNA extraction efficiency is carried out by adding an internal standard bacterial strain Xcc8004. The simple method has simple requirements on sample pretreatment; the quantity of the required environmental samples is small; the obtained DNA has relatively high concentration and purity; the extraction efficiency is relatively high; the analysis and detection of antibiotic resistance gene pollution in the complex environmental samples can be met; and the method is simple and reliable and has practical application value.

The invention discloses a method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs. The method comprises the following steps of: carrying out lysis on a to-be-extracted substance by using guanidinium isothiocyanate lysate; adsorbing RNA by a silica gel membrane; removing impure protein by washing liquor I; removing impurities by washing liquor II; carrying out DEPC (Diethylpyrocarbonate) water-washing to remove nucleic acid, wherein the guanidinium isothiocyanate lysate comprises 3M-7M guanidinium isothiocyanate, 0.6%-1.0% TriTon-100, 30mM-50mM Tris-Cl, 5mM-15mM DTT (DL-Dithiothreitol), 60 mu g/mL-90 mu g/mL protease K, 10mM-30mM EDTA (Ethylene Diamine Tetraacetic Acid), and PH of the guanidinium isothiocyanate lysate is 4.3-4.6; the washing liquor I comprises 5M-6M guanidine hydrochloride, 53%-59% absolute ethyl alcohol, 70-90 mu g/mL protease K, and PH of the washing liquor I is 6.4-6.6; the washing liquor II comprises 70%-80% alcohol. The method disclosed by the invention has the advantages of being simple in extracting process, short in period, low in cost, and capable of simultaneously extracting RNA virus and DNA virus nucleic acid in an animal blood serum sample and a double-swab sample.

The invention belongs to the field of biological products, and relates to a detection kit and a detection method for rapidly detecting salmonella by utilizing an LAMP (loop-mediated isothermal amplification) technique. The kit is composed of a salmonella genome extracting reagent and an LAMP reaction reagent, wherein the salmonella genome extracting reagent comprises the following components: SDS (sodium dodecyl sulfate), protease K, CTAB (cetyltrimethyl ammonium bromide)/NaCl solution and NaAC; and the LAMP reaction reagent is composed of a reaction liquid A and a reaction liquid B, the reaction liquid A comprises the following components: 10*Lampbuffer, Bst DNA (deoxyribonucleic acid) polymerase, dNTP (deoxy-ribonucleoside triphosphate), an interprimer 1, an interprimer 2, an outer primer 1, an outer primer 2, lycine and ultrapure water, and the reaction liquid B is a fluorescent dye. The invention can be used to carry out LAMP detection on salmonella, has the characteristics of rapid detection, high accuracy, high sensitivity and convenient field application, and can be widely applied in the fields of veterinarian, food and exit-entry quarantine, etc.

The invention relates to an extraction method of total DNA of microorganisms from a soil sample. The extraction method particularly includes following steps: (1) adding a DNA extraction buffer solution to the soil sample and performing repetitive freeze-thawing in liquid nitrogen and hot water bath; (2) adding a protease K, performing a water bath treatment process at 37 DEG C for 30 min; (3) adding a sodium dodecyl solution, performing a water bath treatment process at 65 DEG C for 30 min; (4) performing centrifugation to remove precipitations and collect a supernate; (5) performing extraction with a mixed solution containing phenol, chloroform and isoamylol and performing centrifugation to collect a supernate; (6) performing extraction with a mixed solution containing chloroform and isoamylol and performing centrifugation to collect a supernate; (7) adding isopropanol to the supernate in the step (6) to performing precipitation and collecting a precipitation after centrifugation; (8) adding ethanol for washing the precipitation and drying the precipitation at the room temperature; (9) adding a TE buffer solution for dissolving the precipitation to obtain a DNA crude extraction; and (10) purifying the DNA crude extraction through an adsorption column, packaging the purified DNA crude extraction and storing the purified DNA crude extraction at -20 DEG C. The extraction method is especially effective when being used in samples from deep soil and dry sand which are less in biomass and in a clayed soil sample. The DNA extraction can be directly used in subsequent molecular biological detection.

The invention discloses a method for determining a selenium form in a selenium-rich egg, which is characterized by comprising the following steps: 1) pretreating the selenium-rich egg, using an extraction buffer Tris-HCl for extracting a sample of the selenium-rich egg, adding lipase for extracting if the selected selenium-rich sample is a yolk sample, then successively adding protease K and protease XIV with an effective amount for hydrolysis extraction to obtain a sample solution to be measured; directly and successively adding protease K and protease XIV with an effective amount hydrolysis extraction to obtain the sample solution to be measured if the selected selenium-rich egg sample is a egg white sample; 2) using combination of hydride generation-atomic fluorescence spectroscopy and high performance liquid chromatography for respectively determining a solution with a standard selenium form and the sample solution to be measured. The method of the invention is simple and easy to operate, the repeatability is good, and the used reagent has the advantages of no toxicity and no pollution.

The invention discloses a salt-tolerant ethanol-tolerant protease-tolerant surfactant-tolerant exoinulinase, a gene thereof, a vector and a strain. The exoinulinase InuAJB13 possesses the following properties: the optimum pH is 5.5, 50% or more of enzymatic activity is maintained in the pH scope of 4.0-7.0; the remnant enzyme activity reaches 90% or more after exoinulinase is processed by a buffer with a concentration of 0.1 M and pH of 4.0-7.0 for 1 h; the optimum temperature is 55 DEG C, and exoinulinase has the enzyme activity at 10-70 DEG C; a NaCl solution with a concentration of 0.6-4.5 M is capable of improving the enzyme activity by 0.2-0.6 times; 100% of the enzyme activity can be kept after exoinulinase is processed by a NaCl solution with a concentration of 0.2-4.5 M at 37 DEG C for 60 min; exoinulinase keeps the activity in 10% (V/V) of ethanol; exoinulinase keeps 88% or more of the activity after being processed in 3.0-15.0% (V/V) ethanol at 37 DEG C for 60 min; exoinulinase activity is not influenced or slightly influenced by trypsin, protease K, surfactants, most of metal ions, and commercial liquid laundry detergents; and exoinulinase is capable of hydrolyzing inulin, cane sugar, raffinose, stachyose, beta-2,6-fructan (levan) and soluble starch. The exoinulinase disclosed by the invention is applicable to industries such as feed, foodstuff, washing and biofuels.

The invention discloses a method for determining the form of selenium in selenium-enriched edible fungi. The method is characterized by comprising the following steps of: (1) pretreating selenium-enriched edible fungi: using deionized water as an extracting solution to extract a crushed selenium-enriched edible fungus sample, then adding effective amounts of protease K and protease XIV for hydrolysis so as to extract a sample solution to be detected; (2) conducting hydride generation: combining atomic fluorescence spectrometry with high performance liquid chromatography to determine a standard selenium form solution and the sample solution to be detected respectively. The method of the invention has the advantages of simplicity and easy operation, good repeatability, and employment of non-toxic and pollution-free reagents.

The invention discloses a virus nucleic acid extraction kit. The virus nucleic acid extraction kit comprises a lysis solution, a first rinsing solution, a second rinsing solution, an elution solution,protease K and magnetic beads; the lysis solution comprises sodium perchlorate with the concentration of 1.0-5.0 mol/L, Tris-HCl with the concentration of 10-200 mmol/L, EDTA with the concentration of 1-50 mmol/L, and Triton X-100 with the concentration of 50-100 microlitre/milliliter; the first rinsing solution is sodium perchlorate solution with the concentration of 2.0-2.5 mol/L, and the solvent of the sodium perchlorate solution is 70-80% ethyl alcohol; the second rinsing solution comprises DEPC water and absolute ethyl alcohol, wherein the volume ratio of the DEPC water to the absolute ethyl alcohol is (3-4):(6-7); the elution solution is Tris-HCl-DEPC water solution with the concentration of 8-12 mmol/L. The invention further discloses a method for extracting viral nucleic acid by using the extraction kit. Through optimization of the reagent formula, the extraction kit has the advantages of being rapid and efficient and avoiding pollution, a large amount of time and reagent canbe saved, and the extraction efficiency of the virus nucleic acid is improved.