36 results about "Dna recovery" patented technology

The present invention relates to a high sensitivity crime scene swab device for maximal recovery of trace forensic DNA evidence left at a crime scene for DNA PCR analysis. More particularly, DNA recovery is obtained from fingerprints lifted from surfaces at the crime scene. The invention also relates to a high sensitivity method for DNA analysis of trace DNA obtained by generating small tandem repeat (STR) profiles using a polymerase chain reaction protocol.

The invention relates to a microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora. Before cell lysis, the method performs pretreatment on a sample and separates microbial cells from the excrement sample to avoid the problems that pollutants are difficult to remove and the DNA recovery rate is low in a purification step. According to the invention, phenol and chloroform are not used in the extraction process, thus harm to the physical health of experimenters is reduced. The OD260 / OD230 and OD260 / OD280 of the extracted intestinal microorganism DNA are approximate to standard values, and the intestinal microorganism DNA can be directly applied to molecular operation to evaluate the diversity of animal intestinal microflora.

The invention discloses a soil DNA extracting method for evaluating diversity of a microbial community of a plant root system. In the method, pretreatment is carried out on a sample before cell splitting, extracellular DNA and humic substances are removed, and the problems of difficult removal of the humic substances and low DNA recovery rate existing in a purification step are solved. In the extraction process of the method, phenol or chloroform is not used so as to reduce harm on the health of experimenters, obtain complete DNA and molecular fragments greater than 10kb and achieve high yield. OD260 / OD230 and OD260 / OD280 of the extracted soil microorganism DNA are close to standard values and can be directly applied to molecular operation so as to evaluate the diversity of the microbial community of the plant root system.

The invention relates to an agent formula used for collecting and recovering trace amount of DNA by a one-step method and a corresponding kit. The agent formula is mainly characterized in that the agent which is used for collecting and recovering trace amount of DNA and consists of a pH buffer system, salt ions, a surfactant, chelate resin and prolease is adopted, can furthest recover spot forensic material evidence DNA and has good effect on the recovery of sweat fingerprint DNA residual on different materials. The collected DNA can be directly used for the subsequent analysis without the steps of DNA extraction and purification. Compared with the traditional methods, the method has the characteristics of high DNA recovery ratio, simple and convenient operation, low cost, high accuracy and good repeatability, and the developed kit based on the formula can be directly used for collecting DNA samples of the crime scene in the first time.

The present invention relates to a high sensitivity crime scene swab device for maximal recovery of trace forensic DNA evidence left at a crime scene for DNA PCR analysis. More particularly, DNA recovery is obtained from fingerprints lifted from surfaces at the crime scene. The invention also relates to a high sensitivity method for DNA analysis of trace DNA obtained by generating small tandem repeat (STR) profiles using a polymerase chain reaction protocol.

A beta-agaropectinase AgaXa is a novel agaropectinase coded by an agaropectinase gene agaXa of Catenivulumsp.X3. The beta-agaropectinase AgaXa is special by having stable enzyme activity and high enzyme vitality at 47 DEG C (solidification temperature of agarose is 40 DEG C). Therefore, the invention has advantage to be applied to DNA recovery from agarose gel. The beta-agaropectinase AgaXa of the invention can specifically degrade agarose to generate neoagaro-oligosaccharides and can be applied to production of polyoligosaccharides. An escherichia coli recombinant bacterial strain BL21-pET32-agaXa of the invention can express a large quantity of the beta-agaropectinase AgaXa, is easy for purification and can be applied to production of beta-agaropectinase AgaXa, with a low cost.

The invention relates to the technical field of molecular biology and particularly relates to a magnetic bead reagent applied to screening super-long fragment nucleic acids and a screening method. Thereagent comprises silicon oxide hydroxyl magnetic beads with the concentration of 20-800ng / uL, Tris-HCl with the concentration of 5-50mmol / L, sodium chloride with the concentration of 1-60mol / L, sodium laurate with the concentration of 0.1-20mol / L, guanidine hydrochloride with the concentration of 0.1-10mol / L, NaN3 with the concentration of 0.1-100mol / L, polyethylene glycol with the volume percent concentration of 1-25% and EDTA (Ethylene Diamine Tetraacetic Acid) with the pH value of 8 and the concentration of 0.1-10mmol / L. The reagent aims at the problem in the prior art that the large-fragment DNA recovery effect is poor in the high-throughput sequencing library construction process, large fragments are separated and screened by using magnetic beads by designing and optimizing the buffer solution of the magnetic beads, the magnetic bead reagent is particularly applicable to the long-fragment single-molecule sequencing of PacBio, Nanopore and the like, the fragment length of the sequencing data can be greatly improved, and better sequencing results can be obtained.

The invention discloses an improved adenovirus vector system and a virus preparation method. The system takes a pDONR221 vector as an initial vector to make improvement, and multiple cloning sites and an IRES-GFP fragment are inserted. The system combines enzyme digestion connection and recombination reaction technologies, can efficiently construct a vector, and also reduces the reagent cost. The invention also discloses an improved method of purification following after enzyme digestion of the virus vector. The method abandons traditional purification methods adopting phenol, chloroform and isoamyl alcohol, and uses a conventional DNA recovery column or direct heat inactivation method. The method is simple and feasible, shortens the operation time, reduces the material cost, and has no need for toxic phenol and chloroform reagents. The adenovirus system combining the two aspects has improved preparation efficiency and reduced cost, and still maintains high efficiency in infected cells.

The invention belongs to the technical field of molecular biology, and particularly relates to a paramagnetic particle method for extracting residual DNAs (Deoxyribose Nucleic Acids) from a recombinant protein product. The invention discloses a paramagnetic particle method for extracting residual DNAs (Deoxyribose Nucleic Acids) from a recombinant protein product. The method comprises a sample preparing step, an enzyme digestion step, a DNA combining step, a DNA cleaning step and a DNA eluting step, and is characterized in that in the enzyme digestion step, the mass ratio of protease K to proteins in the recombinant protein product is at least 1:6, and incubation is performed at the temperature of 37 DEG C for not less than 10 hours; and in the DNA eluting step, a water bath is performed at the temperature of 70 DEG C for not less than 10 minutes. Compared with the conventional method, the method has the advantages that the DNA recovery rate can be raised to over 50 percent, the recovery rate of a trace quantity of DNAs (less than 1pg) can also exceed 70 percent and can be stabilized between 70 percent and 130 percent, and relevant statutable requirements are met. The method is suitable for extracting residual DNAs from an intermediate and a stock solution in the preparation process of the recombinant protein product, can be used for monitoring the extraneous DNA removing capability of a purifying process, and meets the batch inspection requirement of residual DNAs in the stock solution; and an effective method is provided for the extraction of residual DNAs from the recombinant protein product.

The invention discloses a construction method of a recombinant xylanase XynB with an anti-inhibition activity. The method comprises the steps that whole genome of wheat is extracted, following specific primers are used to amplify active site sequences in third exons of XynB genes of the wheat, gel is excised for DNA recovery by electrophoresis after the DNA fragments are digested with restrictionendonucleases EcoR V and EcoR I and construct recombinant expression vectors with a carrier pET30a+, a recombinant plasmid pET30a-Xyn B is obtained after transformation by transformed bacteria, the recombinant plasmid pET30a-Xyn B is transformed into engineering bacteria, monoclonal colonies are obtained after culturing and transferred to a culture medium for culturing and inducing protein expression to obtain recombinant xylanase XynB; wherein the specific primers are: forward primers: 5'-TC (u) GATATC ( / u) ATGCAGCTGGACAACGCCTT-3', reverse primers: 5'-TC (u) GAATTC ( / u) TGCGTCCGTCTTCCACTCCC-3'. The obtained recombinant xylanase XynB has a strong activity, and unpurified enzyme solution is taken to detect DNS. Birchwood xylan is used as a substrate to calculate, and a specific activity ofthe recombinant xylanase Xyn B can reach to 67.30U / mg.

The present invention discloses an extraction reagent and an extraction method for fermented bean curd microorganism flora DNA. The extraction reagent comprises a cell separation solution, a cell rinsing solution, a cell lysing solution, a PCR inhibitory substance precipitation solution and a washing solution. Based on characteristics of high content of soybean residues and richness of organic acids and amino acids in fermented bean curd samples, processes of fermented bean curd microorganism recovery, cell lysis, PCR reaction substance removal and highly-efficient DNA recovery are designed. For the PCR reaction substance removal, a combination method of ammonium sulfate, guanidine hydrochloride and CTAB is applied, and at the same time, polysaccharides, polyphenols and interfering proteins are removed. Therefore, high-quality DNA is acquired for subsequent molecular biology experiments. The method can simultaneously extract DNA of fermented bean curd bacteria and fungi, and the extracted DNA is successfully used for PCR reaction and high-throughput sequencing of 16S rRNA and ITS genes and is also successfully applied to sequencing and analysis of metagenomics.

The present invention provides a novel method to fabricate silica nanostructures on thin polymer films based on silica deposition and self-wrinkling induced by thermal shrinkage. These micro- and nano-scale structures have vastly enlarged the specific area of silica, thus the silica nanomembranes can be used for solid phase extraction of nucleic acids. The inventive silica nanomembranes are suitable for nucleic acid purification and isolation and demonstrated better performance than commercial particles in terms of DNA recovery yield and integrity. In addition, the silica nanomembranes have extremely high nucleic acid capacity due to its significantly enlarged specific surface area of silica. Methods of use and devices comprising the silica nanomembranes are also provided.

The invention discloses a sol buffer for gel DNA recovery, a kit for gel DNA recovery and an application method of the kit. The sol buffer is prepared from guanidinium isothiocyanate and NaCl, and preferably from 3-3.2M guanidinium isothiocyanate and 1.5-3M NaCl. The invention also discloses a preparation method of a DNA specific adsorption material matching the sol buffer, and a kit for gel DNA recovery and an application method of the kit. The sol buffer for gel DNA recovery and the matching DNA specific adsorption material are of low cost, the preparation is simple and quick (about 2 hours), the application is easy, the effect is good, and the recovery rate is as high as 50% and above; simply by adopting conventional instrument and reagents of a laboratory, the initial amount of singleDNA recovery can reach 200mu g and above, and the recovered DNA can be applied to follow-up PCR, sequencing, etc.

The invention provides a method for extracting a grape genome DNA from wine. The method comprises the following steps: 1) DNA enrichment; 2) removal of polysaccharides; 3) crude extraction of DNA; 4) DNA purification; and 5) DNA recovery, thus obtaining the grape genome DNA. The DNA solution extracted by adopting the method is clear, the extraction and purification efficiency is high, and the obtained DNA has high concentration, good purity and good integrity.

The invention provides a use of agarose in DNA extraction. In DNA extraction, agarose is used. The invention further provides a DNA extraction kit and a DNA extraction method. The DNA extraction kit comprises a box and an individually stored reagent in the box. The reagent comprises agarose. The DNA extraction method comprises adding agarose in DNA extraction. An experiment proves that through use of agarose in DNA extraction, a recovery rate of a trace amount of DNA in extraction is improved and good PCR amplification effects and a complete STR parting result are obtained. Compared with the conventional extraction methods such as a Chelex-100 method and a paramagnetic particle method, the DNA extraction method provided by the invention has a higher DNA recovery rate.

The invention relates to the method used to reclaim DNA from polyacrylamide glue after electrophoresis. It includes the following steps: washing the adhesive tape sample by dH2O, rubbing into powdery by gel rubbing stick, gel soaking, gel removing, DNA connecting, DNA absorbing, DNA washing, DNA elution. Its the advantages are that it has large DNA recovery quantity, high purity, short operation time, simple operation, no need for special device, lost cost; and it can be used in kit manufacturing and agarose gel DNA recovery.

The invention discloses a soil DNA indirect extraction method for evaluating the diversity of a plant root system microflora. Because the mean density of bacteria is far smaller than the mean density of soil minerals, a sample is pretreated before cell lysis, and microbial cells are separated from the soil sample, thereby avoiding the problems of difficult humus removal and low DNA recovery rate occurring in the purification process. In the extraction process, the invention does not use phenols or chloroform, the damage to the health of an experimenter is reduced, the obtained DNA is complete, a molecular segment is larger than 10kb, and the yield is high. The OD260 / OD230 and the OD260 / OD280 of the extracted soil microbe DNA approach to a standard value, and the soil DNA indirect extraction method can be directly applied to molecular operation so as to evaluate the diversity of the plant root system microflora.

Described herein are methods and means for reliably releasing maternal and / or paternal DNA from single seeds without substantially affecting the subsequent germination of the seeds. The methods are particularly suited for genome analysis of small grain cereals, including wheat. The methods may include enzymatic treatments for improved DNA recovery.

The invention discloses a construction method of a drug screening system for treating senile valvular heart disease by using a single traditional Chinese medicine. The construction method specifically comprises the following steps of constructing pmirGLO-RAGE5'-UTR and pmirGLO-mut-RAGE vectors; annealing the target fragment of the gene to synthesize double-stranded DNA, performing thermal denaturation at 95 DEG C for 2 minutes, lowering the temperature by 1 DEG C every 90 seconds to 25 DEG C, standing for 30 minutes, storing at 4 DEG C, and performing cryopreservation at -20 DEG C for later use; carrying out double enzyme digestion on the pmir GLO vector by using Sac I and Xho I enzymes so as to unlink the pmir GLO vector to be linearized; carrying out DNA recovery on a double-enzyme digestion product through agarose gel electrophoresis, and connecting the linear vector with the RAGE5 '-UTR vector; connecting the mutant target fragments by the same method to construct mutant recombinant vector plasmids; transforming the two recombinant vector plasmids and the no-load plasmid into escherichia coli DH5 alpha, picking bacterial colonies, and extracting and identifying the plasmids; and culturing 293T cells, transferring the 293T cells into plasmids containing recombinant pmirGLO-RAGE5'-UTR or pmirGLO-mut-RAGE through LipofectamineTM2000, transfecting for 24 hours, detecting the transfection efficiency, and selecting stably transfected cells, thereby successfully constructing the screening system. The screening system is simple and effective, and target sites can be replaced for popularization and application.

The present invention provides a detection method of a transformed DNA recovery rate. The detection method comprises the following steps: 1) qPCR is conducted on a DNA sample with primers to obtain atransformed Ct value, wherein the DNA sample is transformed to convert an unmodified cytosine base to a uracil base, and the primers recognize a cytosine base-free DNA fragment in the DNA sample; and2) the Ct value and the primers are used to conduct qPCR of an untransformed DNA sample and the obtained Ct value is used to calculate a DNA recovery rate. The method further comprises conducting qPCRon the transformed DNA sample with the DNA primers recognizing the transformed DNA to obtain a Ct value and then evaluating the DNA recovery rate in the step 2).

The invention belongs to the technical field of molecular biology, and particularly relates to a paramagnetic particle method for extracting residual DNAs (Deoxyribose Nucleic Acids) from a recombinant protein product. The invention discloses a paramagnetic particle method for extracting residual DNAs (Deoxyribose Nucleic Acids) from a recombinant protein product. The method comprises a sample preparing step, an enzyme digestion step, a DNA combining step, a DNA cleaning step and a DNA eluting step, and is characterized in that in the enzyme digestion step, the mass ratio of protease K to proteins in the recombinant protein product is at least 1:6, and incubation is performed at the temperature of 37 DEG C for not less than 10 hours; and in the DNA eluting step, a water bath is performed at the temperature of 70 DEG C for not less than 10 minutes. Compared with the conventional method, the method has the advantages that the DNA recovery rate can be raised to over 50 percent, the recovery rate of a trace quantity of DNAs (less than 1pg) can also exceed 70 percent and can be stabilized between 70 percent and 130 percent, and relevant statutable requirements are met. The method is suitable for extracting residual DNAs from an intermediate and a stock solution in the preparation process of the recombinant protein product, can be used for monitoring the extraneous DNA removing capability of a purifying process, and meets the batch inspection requirement of residual DNAs in the stock solution; and an effective method is provided for the extraction of residual DNAs from the recombinant protein product.

The invention relates to an agent formula used for collecting and recovering trace amount of DNA by a one-step method and a corresponding kit. The agent formula is mainly characterized in that the agent which is used for collecting and recovering trace amount of DNA and consists of a pH buffer system, salt ions, a surfactant, chelate resin and prolease is adopted, can furthest recover spot forensic material evidence DNA and has good effect on the recovery of sweat fingerprint DNA residual on different materials. The collected DNA can be directly used for the subsequent analysis without the steps of DNA extraction and purification. Compared with the traditional methods, the method has the characteristics of high DNA recovery ratio, simple and convenient operation, low cost, high accuracy and good repeatability, and the developed kit based on the formula can be directly used for collecting DNA samples of the crime scene in the first time.

The invention discloses a method for quickly and efficiently purifying and recovering small fragment DNA by using a homogenizer to grind gel. Grind the gel containing small fragments of DNA in TE buffer directly with a homogenizer, collect the supernatant by centrifugation, precipitate the DNA under the action of salt ions and absolute ethanol, and redissolve the precipitated DNA in TE buffer. So as to achieve efficient purification and recovery of small fragments of DNA. The method has simple operation steps and improves recovery efficiency. At the same time, the method provides a new means for the purification and recovery of small and medium molecular weight DNA for research and application such as gene cloning, mutation detection, gene diagnosis and therapy.

The Bacillus cellulase gene FIB1X suitable for rice straw returning to the field belongs to the field of farming and cultivation technology. The cycle of using bacteria to treat straw is very short, usually it can be completed in a few days, so that air pollution caused by burning straw can be avoided. Various problems caused by other processing methods. Therefore, the hydrolysis circle experiment to measure the cellulase activity of all intestinal bacteria of Brachypterus sp. was found to have the strongest cellulase activity. Then, through the PCR amplification of the gene, DNA recovery, ligation reaction, E. coli heat shock transformation, E. coli plasmid DNA extraction, gene induction expression, protein electrophoresis detection, sequence determination and analysis, the Bacillus cellulase gene FIB1X was obtained. The nucleic acid sequence is Seq ID No: 1. Finally, the enzyme activity of the protein expressed by the Bacillus cellulase gene FIB1X was determined, and the result showed that the enzyme activity was relatively high.

The invention discloses a method for detecting an MLL-AF4 fusion gene. The method sequentially comprises the following steps: 1, acquiring sample DNA, 2, preparing a PCR reaction liquid, wherein a primer used for amplifying the MLL-AF4 fusion gene is F: 5'-CCGCCCAAGTATCCCTGTAAAC-3', and a primer used for amplifying the MLL-AF4 fusion gene is R: 3'-TCGTTATGTATGCGGATCATGTCG-5', 3, carrying out target strip amplification by taking the DNA of a to-be-detected sample as a template, and amplifying a required DNA fragment through the designed primers, 4, carrying out target DNA recovery by carrying out 1% agarose gel electrophoresis on the PCR reaction solution, and recovering a target strip, 5, purifying the DNA fragments obtained in the step 1, 6, carrying out conversion on the purified DNA, and 7, carrying out sequencing, and judging whether the fusion gene is the MLL-AF4 fusion gene or not. According to the method for detecting the MLL-AF4 fusion gene, primers suitable for PCR amplification are designed, high specificity, accuracy and sensitivity are achieved in the detection process, a target band is amplified more rapidly and efficiently, and repeatability is achieved.

The invention provides a kit for recovering DNA (Deoxyribose Nucleic Acid) by using agarose gel through a column method. The kit comprises a sol solution, a DNA adsorption column and a rinsing solution, the sol solution consists of guanidine isothiocyanate with the concentration of 4.1 mol / L to 4.8 mol / L, an acetic acid-potassium acetate buffer solution with the concentration of 0.1 mol / L to 0.5 mol / L and phenol red with the concentration of 0.01 g / L to 0.05 g / L; a DNA adsorption column adopts a three-layer silica gel membrane column; the rinsing liquid is prepared from 0.1 mol / L to 0.5 mol / L of NaCl, 0.001 mol / L to 0.005 mol / L of EDTA (Ethylene Diamine Tetraacetic Acid), 0.01 mol / L to 0.05 mol / L of a Tris-HCl buffer solution and 75 percent of absolute ethyl alcohol. The invention also provides a method for purifying and recovering DNA by using the kit for recovering DNA by using the column method agarose gel. The agarose gel DNA recovery kit and the method for purifying and recovering DNA by using the kit have the advantages of low cost, stability and high efficiency.

The invention discloses an improved adenovirus vector system and a virus preparation method. The system takes a pDONR221 vector as an initial vector to make improvement, and multiple cloning sites and an IRES-GFP fragment are inserted. The system combines enzyme digestion connection and recombination reaction technologies, can efficiently construct a vector, and also reduces the reagent cost. The invention also discloses an improved method of purification following after enzyme digestion of the virus vector. The method abandons traditional purification methods adopting phenol, chloroform and isoamyl alcohol, and uses a conventional DNA recovery column or direct heat inactivation method. The method is simple and feasible, shortens the operation time, reduces the material cost, and has no need for toxic phenol and chloroform reagents. The adenovirus system combining the two aspects has improved preparation efficiency and reduced cost, and still maintains high efficiency in infected cells.

It is intended to provide a method for DNA recovery capable of more reliably recovering a cDNA fragment of interest from a group of cDNA fragments in which various kinds of cDNA fragments are present. The method for DNA recovery comprises a first PCR step for carrying out a PCR reaction on the group of cDNA fragments each having a first adaptor sequence on one end and a second adaptor sequence on the other end, using a first primer having a sequence complementary to the first adaptor sequence and also having a labeling substance and a second primer having a sequence complementary to the second adaptor sequence. Also, an electrophoretic step for carrying out a gel electrophoresis on the group of cDNA fragments amplified in the first PCR step and, on the basis of a result of the electrophoretic step, a recovery step for cutting out a gel portion containing a cDNA fragment of interest from a gel and then recovering the cDNA fragment from the gel are comprised.

The invention discloses a sperm chromatin co-immunoprecipitation method. The method belongs to the field of target nucleic acid purification and next-generation sequencing library building sample pretreatment. The method includes the following steps: sperm digestion: subjecting the sperms to paraformaldehyde immobilization, cleaning and split, digesting the sperms by adopting a micrococcus nuclease digestion-ultrasonic combined method to obtain digestive juice; ultrasonic parameters are as follows: performing ultrasonic treatment for 30 seconds by the interval of 45 seconds for 10 cycles; setting the digestion parameters of the micrococcus nuclease are as follows: the enzyme addition amount is 200U / 106 sperms, the enzymolysis temperature is 37 DEG C, and the enzymolysis time is 30 minutes; chromatin separation: performing preliminary cleaning on the digestive juice to obtain chromatin; and chromatin co-immunoprecipitation: forming a compound by the antibody and protein A agrose, combining the compound with a corresponding antigen in chromatin, and then cleaning and eluting to obtain a co-immunoprecipitation reaction solution. According to the method, ultrasonic and enzyme digestion are combined, so that the digestion efficiency is improved, the DNA recovery rate is increased, the library building difficulty is reduced, and the sperm co-immunoprecipitation efficiency is improved.

The invention relates to an agarose gel electrophoresis product recovering method, which prevents experimenters from touching gel repeatedly, reduces rate of contamination of DNA (deoxyribonucleic acid), improves DNA recovery efficiency, shortens time of gel exposed under ultraviolet light, and reduces subsequent experiment risks caused by mutagenesis and damages of nucleic acid samples during ultraviolet excitation. A device utilizing the agarose gel electrophoresis product recovering method is unique in design, easy in manufacturing process, skillful, portable, simple and convenient in use and low in price.