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Cell Subculture Service

Introduction Cell subculture is a cell or microbiological culture method made by transferring some or all cells from a previous culture to a new vessel with a fresh growth medium to provide more room for continued growth. Depending on the properties of the cells, there are two different ways for cell subculture: 1. For non-adherent cell culture, the cells are only grown in solution but won’t attach to the surface, these cells can be subcultured by simply taking a small volume of the primary culture and diluting it in fresh growth medium. 2. For adherent cell culture, epically the culture of many mammalian cell lines, the cells would attach to a surface such as the bottom of the culture flask. These cell types have to be detached from the surface by the trypsin before they can be subcultured. The procedures are like below: Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get stability, and it means a reliable partner to h...

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Genome Walking

Introduction Genome walking (GW) is a molecular procedure for the direct identification of nucleotide sequences from purified genomes, the only thing that we need to know is the nucleotide sequence from which to start. There are numerous methods for GW, and they can be classified into three main categories: (1). Restriction-based GW methods: it’s requiring a restriction digestion of genomic DNA and ligation of restriction fragments to DNA-cassettes; (2). Primer-based GW methods: the PCR amplification in this method are directly carried out using a variously designed combinatorial coupled to a sequence specific primer; (3). Extension-based GW methods: the extension of a sequence specific primer and subsequent 3’-tailing of the resulting single strand DNA (ssDNA) provide the substrate for the final PCR amplification (Fig. 1). Fig. 1 Main GW strategies Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get stability, and it means a reliable partner to help streamline your R&D pro...

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Site-directed Mutagenesis Service

Introduction Side-directed Mutagenesis (SDM) is an in vitro procedure that uses custom designed oligonucleotide primers to confer a specific and intentional mutation in a double-stranded plasmid DNA. The SDM is a very useful tool for gene research, it could be used to study changes in protein activity that occur as a result of the DNA manipulation; to select or screen for mutations that have a desired property, or to introduce or remove restriction endonuclease site or tags. Generally, we introduce site-directed mutagenesis through PCR method, including the insertion, deletion and point mutation. The procedures are like below: 1. Design a primer which contains the desired change, which could be based substitution, addition or deletion. 2. Perform PCR: During PCR, the mutation is incorporated into the amplicon, replacing the original sequence. Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get stability, and it means a reliable partner to ...

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Microsatellite Analysis

Introduction Microsatellite, also called Short Tandem Repeats (STRs) or Single Sequence Repeats (SSRs), is a genetic marker that can be used to locate a specific segment of genetic material that has a known location on a chromosome. Microsatellites are codominant in nature, highly polymorphic, easily typed, and Mendelian inherited, these properties made them very suitable for the study of population structure, pedigree analysis and detect differences among closely related species. The major advantages of microsatellite markers are short size range, uninterrupted stretches of identical repeat units, the high proportion of polymorphisms, insights gained in understanding the mutational process, etc. These advantages help to develop statistical procedures for interpopulation comparisons and made micorsatellite marker be a widely used marker for high-resolution population analysis. Procedures: 1. Collect microsatellite sequence information from resource species (closely related to or from the same family), design primers. 2. Genomic DNA isolation from species of interest. 3. PCR amplification of DNA using resource primers. 4. Gel electrophore...

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DNA Methylation Analysis

Introduction Methylation is a normally occurring modification to DNA. It is characterized by the biochemical addition of a methyl group to the cytosine 5-carbon in cytosine-phosphate-guanosine (CpG) dinucleotides via a methytransferase enzyme. DNA methylation plays an important role in regulating gene expression. Aberrant DNA methylation has been implicated in many disease processes, including cancer, obesity, and addiction. It’s also a common subject of agrigenomic investigations into responses to drought, temperature extremes, and other environmental changes. Fig. 1 The reaction of DNA methylation High-throughput technologies such as next-generation sequencing (NGS) and microarrays enable researchers to perform genome-wide methylation profiling, and researchers can also use high-performance capillary electrophoresis and methylation-sensitive arbitrarily primed PCR to quantify DNA methylation. For gene-specific methylation analysis, the most popular method is the bisulfite reaction-based method, this method use sodium bisulfite to convert the unmethylated cytosines on DNA to uracils, thus the modified DNA template ...

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SNP/CNV/AFLP Genotyping Service

Introduction Genotyping is the process of determining which genetic variants an individual possesses, it can be performed through a variety of different methods, depending on the variants of interest and the resources available. SNP Genotyping Analysis A single-nucleotide polymorphism (SNP) is sequence variation at the single-base level. They can be found in coding, non-coding, and intronic regions of genomes, and they may affect transcription factor binding, gene splicing, protein folding and many other elements at the gene and transcript level. Fig. 1 SNP Genotyping methods There are several methods for SNP genotyping, including dynamic allele-specific hybridization, Molecular bacons, PCR based methods, oligonucleotide ligation assay, etc. Typically, the genotyping protocols start with target amplification and follow with allelic discrimination and production or identification, the allelic discrimination reaction step including primer extension, pyrosequencing, ligation, structure specific cleavage and hybridization, some or all of these steps could be combined and processed in parallel. The last...

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ChIRP Assay

Introduction ChIRP, also called Chromatin Isolation by RNA Purification, is a method based on affinity capture of target IncRNA (long non-coding RNA)-Chromatin complex, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. There are many advantages of ChIRP assay, first, it could identify the binding sites anywhere on the genome, second, it enables the discovery of new binding sites, last but not least, it allows the selection of specific RNA. ChIRP is applicable to many IncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA’s structure or functional domains. Procedures: 1. Chromatin was cross-linked to lncRNA: protein adducts in vivo. 2. Biotinylated tiling probes were hybridized to target lncRNA, and the complexes are purified using magnetic streptavidin beads. 3. The lncRNA bound DNA or proteins was eluted by a cocktail of RNase A and H. 4. Extracted DNA fraction from ChIRP samples was identified by sequencing or quantitate by qPCR. ...

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DNA pull down Assay

Introduction Similar to RNA pull-down assay, DNA pull-down assay is an in vitro method used to purify DNA-binding proteins from tissue and cell samples. Streptavidin beads can bind to biotin-labeled DNA/protein complexes. Centrifuge to separate the conjugate from the supernatant or discard the supernatant, then wash the beads. The proteins in the separation solution were separated by SDS-PAGE and the SDS-PAGE was stained with high sensitivity coomassie brilliant blue. The final step is to cut the desired protein bands from the stained gel for MS to confirm the protein. Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get stability, and it means a reliable partner to help streamline your R&D process. Working with us, you will get the guaranteed service to accommodate your requirements. · Vigorous quality control system to ensure the required quality and reproducibility · Competitive price with fast turnaround time ...

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RNA pull down Assay

Introduction RNA pull-down assay is a technique used to detect the RNA-protein interactions using immunoprecipitation (IP). This technique is commonly used to either confirm the presence of a scant protein or enrich the population of a particular protein, as well as to identify natural binding partners for the captured protein. Biotinylated RNA-pulldown is a useful pulldown Method which theoretically works for all RNA-binding proteins (RBPs), as this assay is performed in a cell-free system. The first step of this method is to synthesis RNAs of interest in the presence of biotinylated cytidine triphosphate (CTP); the RNA then incubated with a cell-free system to allow RBPs to recognize RNA regions to which it has an affinity, while other regions won’t interact with RBPs. After binding complete, the biotinylate tagged RNA is pulled down using streptavidin-coated beads and the RBPs are typically detected by Western blot analysis. Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible....

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Luciferase Reporter Assay

Introduction The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. In this assay, the light could be generated by one of a family of enzymes known as luciferases, and another essential tool in this study is the reporter gene, as they are designed to function as surrogates for genes involved in key cellular process and disease. A good reporter gene includes an easily measurable phenotype, low background activity in the cell under investigation, a large dynamic range, and high reliability and sensitivity. There are a number of genes that could meet these requirements, including members of the family of luciferases, β-galactosidase (β-Gal), chloramphenicol acetyltransferase (CAT), etc. Typically, the reporter gene is cloned with a DNA sequence of interest into an expression vector that is then transferred into cells. After transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or enzymatic activity of the reporter protein. Why Leading Biology? At Leading Biology, we custom protein purification des...

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