Este trabalho foi financiado pela National Science Foundation Programa Ambiental Genomics (Grant No. EF-0723707).

1. Extração de células microbianas: <ol><li> Homogeneizar tapetes microbianos com um pilão de moer estéril, misturando bem. Coloque todos os cerca de 30 g (peso úmido) de material mat homogeneizadas em recipiente estéril de liquidificador Waring, adicionar cerca de 100 mL de NaCl 1 M (ou uma concentração específica para amostra em uso), e mistura três vezes em velocidade média por 1 min com resfriamento intermitente em um freezer -20 ° C por 1 min. Transfira a pasta em um frasco de centrífuga de 250 mL e completar o volume vazio restante com 1 M NaCl. Prepare a solução de NaCl em água autoclavada DI e filtro de esterilizá-lo. </li><li> Além disso desalojar as células microbianas a partir da matriz agitando (150 rpm) usando um vortexer (Vortex-Genie R 2) em temperatura ambiente por 30 min. </li><li> Centrifugar a baixa velocidade (500x g) por 15 min a 4 ° C. Gentilmente transferir o sobrenadante para um balão com perturbação mínima para o sedimento. </li><li> Usando o sedimento peletizada, repita os passos de mistura e 1.2 e 1.3 quatro vezes mais com a adição de NaCl fresco para cada pellet de sedimentos no final do passo 1.3. O sobrenadante de cada extração de NaCl é transferido para um balão fresco. </li><li> Combine os sobrenadantes dos 5 celulares extrações e centrifugar em alíquotas de 200 mL em alta velocidade (25.000 xg) por 15 min a 4 ° C. Desprezar o sobrenadante, ressuspender cada pellet celular em 10 mL de hexametafosfato de sódio a 2%, combine pellets e fazer aumentar o volume para 200 mL com hexametafosfato de sódio a 2%. Lave as células por agitação (150 rpm) em temperatura ambiente por 30 min. No final desta etapa você deve ter apenas um tubo de células para prosseguir para a próxima etapa. </li><li> Centrífuga em alta velocidade (25.000 xg) por 15 min a 4 ° C. Desprezar o sobrenadante, ressuspender pellet celular em 200 mL de TE (50 mM EDTA), e lavar as células por agitação (150 rpm) em temperatura ambiente por 10 min. </li><li> Centrífuga em alta velocidade (25.000 xg) por 15 min a 4 ° C, desprezar o sobrenadante, ressuspender em 15 mL de TE (10 mM EDTA), e armazenar de 200 mL alíquotas a -80 ° C até ser necessário para DNA extrações. </li></ol> 2. Extração de DNA e Purificação: <ol><li> Adicionar 200 mL de NaCl 5 M, 200 mL SDS 10%, e de 100 L 14,3 M β-mercaptoetanol para a alíquota de 200 mL de células microbianas. Misture delicadamente por inversão 4-6 vezes. </li><li> Mistura o assunto para 3 rounds de congelamento e descongelamento por submersão em nitrogênio líquido por 2 minutos seguido de descongelamento em banho-maria a 65 ° C por 5 min. Estender finais descongelamento a 10 min. </li><li> Adicionar 200 mL de acetato de potássio 5 M (pH 5,5) e colocar no gelo por 10 min. Centrifugar a 10.000 xg por 10 min a 4 ° C e transferir o sobrenadante para um novo tubo usando uma ponteira de grande furo. Adicionar 3 mL de RNase A e incubar a 37 ° C por 1h. </li><li> Adicionar igual volume de clorofórmio, agitar brevemente por inversão, e centrifugar a 15.000 xg por 10 min em temperatura ambiente. </li><li> Transferir cuidadosamente a camada superior do sobrenadante para um novo tubo usando uma ponteira de grande furo. Evite pipetagem a camada de interface branco, localizado entre as camadas superior e inferior. Repita o procedimento de extração clorofórmio. </li><li> Adicionar um volume igual de refrigerados (a -20 ° C) isopropanol para o sobrenadante e incubar no gelo por 30 min para o DNA precipitado. </li><li> Centrifugar à máxima velocidade (20.800 xg) por 10 min a 4 ° C para pellet de DNA. </li><li> Desprezar o sobrenadante e lavar DNA pellet com 1 mL de refrigerados (a -20 ° C) etanol 70%. </li><li> Centrifugar à máxima velocidade (20.800 xg) por 10 min a 4 ° C, desprezar o sobrenadante, repita lavar etanol 70%, e de ar seco pellet de DNA por 10 min. Repita lavar etanol 70%. </li><li> Ressuspender o DNA em 25 mL TE (10 mM EDTA), quente a 65 ° C por 5 min, se combinam em um tubo se tubos múltiplos têm sido utilizados, e make-up volume de 500 mL com TE. </li><li> Adicionar 500 mL gelada (ou a 4 ° C) 20% de polietileno glicol (PEG) (preparado em 1,2 M NaCl). Misture delicadamente por inversão e incubar no gelo durante 10 min, e centrifugar a velocidade máxima por 10 min a 4 ° C para pellet de DNA. Remover e descartar o sobrenadante e lavar o DNA peletizadas com 1 mL de etanol 70% gelado, ar seco por 10 min, e ressuspender em 30-50 mL TE ou água de grau molecular. Facilitar a ressuspensão de DNA por aquecimento a 65 ° C por 5 min e DNA loja a -80 ° C. </li></ol> 3. Purity DNA, Concentração e Determinação Tamanho: <ol><li> Determinar a qualidade do DNA através da medição da 260/280 A e A 260/230 relações usando um espectrofotômetro 1000 Nanodrop ou quaisquer outros instrumentos apropriados. </li><li> Determinar a concentração de DNA usando Quant-iT dsDNA kit de Ensaio de acordo com as instruções do fabricante. </li><li> Determinar o tamanho do DNA usando pulso eletroforese em gel de campo. Prepare 1% agarose gel com 0,5 X TBE e usar os seguintes parâmetros na Mapper CHEF XA System; tempo de troca inicial de 0,35 s, um tempo de troca final de 7,67 s, e 120 ° incluído ângulo de um gradiente de 6,0 V / cm em um r linearamplificação fator. Mancha gel com 1 x SYBR Green I para 30 min. Carga entre 700-1000 ng DNA total em gel. Ver fig. 3 para uma forma condensada do protocolo completo. </li></ol> Resultados representativos: Extração de células: Extratos microbianos seqüencial celular (sobrenadante) mostraram que a turbidez dos extratos diminui à medida que aumenta o número de extrações. Isto sugere uma redução no número de células após cada extração de células sucessivas. Importante notar aqui é que, como cada etapa de extração de células adicionais proporciona a oportunidade para a introdução de contaminantes, o número de células extrações devem ser minimizados para que o final de pellet celular é representante da comunidade microbiana total. Outras fontes de contaminação foram controlados através da adopção de técnicas de laboratório adequado estéril. Por exemplo, as soluções foram preparadas em água DI autoclavado e esterilizado por filtração. Recipientes foram esterilizados com álcool, autoclavados e tratados sob luz UV e reticulador ultravioleta. Concentração de DNA e determinação de qualidade: O protocolo rendeu cerca de 2 mg de HMW DNA (35-50 kb) (Fig. 4) por grama de amostra tapete com um A 260 / 280 relação de 1,6, e um A proporção de 260/230 0,7 (Tabela 1). Embora a razão A 260 / 230 parece ser baixa, sem inibição de aplicação de base molecular a jusante, como a amplificação do 16S rRNA genes foi observado em um estudo separado 7. É importante notar que o DNA de hipersalinos esteiras tem duas principais fontes de contaminação; EPS e sais. Portanto, é possível que traços destes contaminantes podem estar influenciando a A 260/230 relações, apesar dos esforços tremendos para reduzir seus efeitos sobre as aplicações de fluxo baixo. DNA determinação do tamanho: Pulso de eletroforese em gel de campo emprega um fluxo pulsante atual com interruptores intermitentes direcional, resultando em um esfregaço de DNA, como mostrado na figura 3. Nosso protocolo rendeu um DNA HMW de aproximadamente 35-50 kb. Enquanto alguns tamanho DNA pode ser menor que 30 kb, é importante ter parte do esfregaço acima de 35 kb dado que a clonagem fosmid requer ~ 40 kb de DNA e insere fragmentos maiores de DNA proporcionar maior acesso às vias biossintéticas intacta. Em nossos estudos, o esfregaço DNA acima de 35 kb foi retirado e purificada de grande vetor de inserção clonagem e outras aplicações molecular. <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="Figura 1"/> Figura 1. Microbiana hipersalinos local de amostragem mat (Lagoa Grande) localizado na Eleuthera, nas Bahamas. <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="Figura 2"/> Figura 2. A seção transversal do tapete microbiano hipersalinos utilizados neste estudo. O tapete microbiano foi obtido a partir de uma lagoa hipersalina localizado na Eleuthera, nas Bahamas. <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="Figura 3"/> Figura 3. Representação esquemática dos processos envolvidos na extração de células microbianas, lise celular, extração e purificação do DNA metagenomic. <a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">Clique aqui para ver uma imagem maior.</a> <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="Figura 4"/> Figura 4. Caracterização do peso molecular do DNA extraído usando pulso eletroforese em gel de campo. Pista M é um marcador HMW, e faixas 1 e 2 são réplicas de DNA metagenomic extraído do tapete hipersalinos Eleuthera usando o protocolo acima. <table border="1"><tbody><tr><td> Método de extração </td><td></td><td> Concentração ng / g </td><td> A 260/280 </td><td> A 260/230 </td></tr><tr><td> PEG </td><td> Rep 1 </td><td> 1806,1 </td><td> 1,64 </td><td> 0,76 </td></tr><tr><td></td><td> Rep 2 </td><td> 2010,8 </td><td> 1,61 </td><td> 0,75 </td></tr></tbody></table> Tabela 1. A medição da concentração e qualidade do DNA extraído de mat hipersalinos microbiana.

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Dado que a remoção total de células a partir de amostras mat complexa e altamente diversificada microbiana não é prático, a principal preocupação é o quão bem as células extraídas representar a comunidade microbiana total mat. Em um estudo anterior, PCR-DGGE análise dos genes 16S rRNA microbiana mostrou que as cinco etapas de remoção de células utilizadas neste protocolo de extratos de células que são representativas da comunidade em geral tapete microbiano 7. O número real de extração de...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> β-mercaptoetanol </td><td> Sigma-Aldrich </td><td> M3148 </td><td></td></tr><tr><td> Polietileno glicol 8000 </td><td> Promega </td><td> V3011 </td><td> 20% em 1,2 M NaCl </td></tr><tr><td> Acetato de potássio </td><td> Fisher Scientific </td><td></td><td> Fisher Scientific </td></tr><tr><td> Quant-iT Assay dsDNA kit </td><td> Invitrogen </td><td> Q33130 </td><td></td></tr><tr><td> RNase </td><td> Epicentro </td><td> MRNA092 </td><td></td></tr><tr><td> Cloreto de sódio </td><td> BDH Chemicals </td><td> BDH8014 </td><td> Conc apropriado. </td></tr><tr><td> Dodecilsulfato de sódio </td><td> Fisher Scientific </td><td> 03-500-509 </td><td> 10% em água </td></tr><tr><td> hexametafosfato de sódio </td><td> Merck Química </td><td> SX0583-3 </td><td> 2% da água </td></tr><tr><td> TBE </td><td> Fisher Scientific </td><td> BP1333-1 </td><td></td></tr><tr><td> CHEF Mapper XA Sistema </td><td> Bio-Rad Laboratories </td><td> 170-3670 </td><td></td></tr><tr><td> NanoDrop 1000 espectrofotômetro </td><td> Thermo Scientific </td><td> ND-1000 </td><td></td></tr><tr><td> Vortexer </td><td> Scientific Industries Inc. </td><td></td><td></td></tr><tr><td> Agente de crosslinking ultravioleta </td><td> UVP </td><td></td><td></td></tr><tr><td> Waring Blender </td><td> Waring laboratório </td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

Análise bem sucedida e precisas e interpretação de dados metagenomic é dependente da extração eficiente de alta qualidade, de alto peso molecular DNA comunidade (HMW). No entanto, as amostras de mat ambientais muitas vezes apresentam dificuldades para a obtenção de grandes concentrações de DNA de alta qualidade HMW,. Tapetes microbianos hipersalinos contêm grandes quantidades de substâncias poliméricas extracelulares (EPS) 1 e sais que podem inibir a aplicações a jusante do DNA extraído. Métodos diretos e duras são muitas vezes utilizados na extração de DNA a partir de amostras refratário. Estes métodos são normalmente utilizados, pois o EPS em esteiras, uma matriz adesiva, liga-se 2,3 DNA durante a lise direta. Como resultado de métodos mais duros de extração, o DNA torna-se fragmentado em tamanhos pequenos 4,5,6. O DNA, assim, torna-se inadequado para os grandes inserir clonagem do vetor. A fim de contornar essas limitações, apresentamos uma metodologia melhorada para extrair DNA HMW de boa qualidade e quantidade de tapetes microbianos hipersalinos. Nós empregamos um método indireto envolvendo a separação de células microbianas a partir da matriz mat fundo através de mistura e centrifugação diferencial. A combinação de procedimentos mecânicos e químicos foi usado para extrair e purificar o DNA das células microbianas extraído. Nosso protocolo produz cerca de 2 mg de HMW DNA (35-50 kb) por grama de amostra de esteira, com um A 260 / 280 relação de 1,6. Além disso, a amplificação do 16S rRNA genes 7 sugere que o protocolo é capaz de minimizar ou eliminar os efeitos inibitórios de contaminantes. Nossos resultados fornecem uma metodologia apropriada para a extração de DNA a partir de HMW tapetes microbianos para estudos funcionais metagenomic e pode ser aplicável a outras amostras ambientais a partir do qual a extração de DNA é um desafio.

Watch this Scientific Journal Video about Extraction of High Molecular Weight DNA from Microbial Mats at JoVE.com

Extraction of High Molecular Weight DNA from Microbial Mats

Nós fornecemos um protocolo melhorado para a extração de DNA de alto peso molecular de tapetes microbianos hipersalinos. Células microbianas são separados da matriz mat antes da extração e purificação DNA. Isso aumenta a concentração, qualidade e tamanho do DNA. O protocolo pode ser usado para outras amostras refratário.

Extração de DNA de alto peso molecular de Esteiras Microbianas

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

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19.4K visualizações.  Arnold School of Public Health, University of South Carolina. Nós fornecemos um protocolo melhorado para a extração de DNA de alto peso molecular de tapetes microbianos hipersalinos. Células microbianas são separados da matriz mat antes da extração e purificação DNA. Isso aumenta a concentração, qualidade e tamanho do DNA. O protocolo pode ser usado para outras amostras refratário.

Vídeo: Extraction of High Molecular Weight DNA from Microbial Mats

Biology of Microbial Communities  - Interview

Biologia de comunidades microbianas - Entrevista

Biologia

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

DNA Extraction from 0.22 &amp;mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

Extração de DNA de 0,22 mM Sterivex Filtros e Césio cloreto de centrifugação em gradiente de densidade

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extração de DNA genômico de alto peso molecular de solos e sedimentos

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Extração de DNA de material parafinado Embedded para análises genéticas e epigenéticas

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Um método de extração de DNA híbrido para a Avaliação de comunidades bacterianas Qualitativa e Quantitativa de amostras de produção de aves de capoeira

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucina Agarose Gel Eletroforese: Western Blotting para glicoproteínas alta peso molecular


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Preparação de Núcleos de tecido parafina-encaixados formalina-fixados para RNA e DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Métodos modificados para carregamento de placas de extração de DNA de alto rendimento reduzem o potencial de contaminação

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolamento de miócitos murinos atrial e ventricular de alta qualidade para medidas simultâneas de Ca2+ transitórios e corrente de cálcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Extração em pequena escala de Caenorhabditis elegans DNA genômico

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...