Bu çalışma, Ulusal Bilim Vakfı Çevre Genomik Programı (Hibe No EF-0.723.707) tarafından finanse edildi.

1. Mikrobiyal Hücre Ekstraksiyon: <ol><li> Mikrobiyal paspaslar steril bir taşlama havaneli ile iyice karıştırılarak homojenize. Yaklaşık 1 dakika süreyle homojenize mat malzeme steril kap içine Waring blender bütün 30 g (ıslak ağırlık), yaklaşık 100 mL 1 M NaCl (ya da örnek kullanım için belirli bir konsantrasyon) eklemek, üç kez ve orta hızda karışımı yerleştirin. 1 dakika boyunca -20 ° C derin dondurucuda aralıklı soğutma. Bulamaç 250 ml santrifüj şişeye aktarın ve kalan boş hacim 1 M NaCl ile doldurun. Otoklava DI su NaCl çözeltisi hazırlayın ve sterilize filtre. </li><li> Ayrıca 30 dakika oda sıcaklığında bir vortexer (Vortex-Genie R 2) (150 rpm) sallayarak matris mikrobiyal hücrelerin yerinden. </li><li> 15 dakika 4 ° C düşük hızlı (500x g) Santrifüj Tortu minimum rahatsızlık ile hafifçe bir balona süpernatantı aktarın. </li><li> Pelet tortu kullanarak 1.2 ve 1.3, 1.3 adım sonunda her tortu pelet taze NaCl ilavesi ile dört kez karıştırma ve adımları tekrarlayın. Her NaCl çıkartılmasından süpernatant taze bir balona transfer edilir. </li><li> 15 dakika yüksek hızda (25.000 xg) 200 ml hacimde 5 hücre ekstraksiyon ve santrifüj süpernatantlar birleştirin 4 ° C Süpernatantı atın, 10 ml% 2 sodyum hexametaphosphate her hücre pelletini tekrar süspansiyon, pelet birleştirmek ve% 2 sodyum hexametaphosphate ile 200 mL birimi oluşturacak. 30 dakika oda sıcaklığında (150 rpm) sallayarak hücreleri yıkayın. Bu adımın sonunda bir sonraki adıma geçmek için hücrelerin sadece bir tüp olmalıdır. </li><li> 15 dakika 4 ° C yüksek hızda (25.000 xg) Santrifüj TE (50 mM EDTA), 200 ml supernatant, tekrar süspansiyon hücre pelletini atın ve 10 dakika oda sıcaklığında (150 rpm) sallayarak hücreleri yıkayın. </li><li> 4 az 15 dakika yüksek hızda (25.000 xg) Santrifüj ° C, 15 ml TE (10 mM EDTA) tekrar süspansiyon süpernatant, atmak ve mağaza 200 mcL alikotları -80 ° C kadar DNA ekstraksiyon için gerekli. </li></ol> 2. DNA Ayırma ve Saflaştırma: <ol><li> 200 mcL 5 M NaCl, 200 mcL% 10 SDS ve 200 mcL mikrobiyal hücre kısım 100 mcL 14.3 M β-mercaptoethanol ekleyin. Tersini 4-6 kez hafifçe karıştırın. </li><li> , 2 dakika, 5 dakika boyunca 65 ° C su banyosu içinde çözülme takip sıvı nitrojen içinde daldırarak donma-çözülme 3 tur Konu karışımı. Son 10 dakika çözülme uzatın. </li><li> 200 mcL 5 M potasyum asetat (pH 5.5) ve 10 dakika süreyle buz yer ekleyin. Santrifüj 10,000 xg&#39;de 10 dakika 4 ° C ve geniş çaplı bir pipet kullanarak yeni bir tüp süpernatantı aktarın. 1hr RNaz 37 A ve inkübe ° C 3 mcL ekleyin. </li><li> Eşit miktarda kloroform, inversiyon kısaca sallamak ve oda sıcaklığında 10 dakika 15.000 xg&#39;de santrifüj. </li><li> Süpernatant üst tabakası, geniş çaplı bir pipet kullanarak yeni bir tüp içine dikkatlice aktarın. Üst ve alt katmanları arasında yer alan beyaz arabirim katmanı pipetleme kaçının. Kloroform-çıkarma prosedürünü tekrarlayın. </li><li> Soğutulmuş, eşit hacimde (-20 ° C) çökelti DNA 30 dakika buz üzerinde inkübe supernatant ve izopropanol. </li><li> 4 az 10 dakika süreyle maksimum hızı (20,800 xg) Santrifüj ° C pelet DNA. </li><li> 1 ml soğuk (-20 ° C)% 70 etanol ile süpernatant ve yıkama DNA pelet atın. </li><li> Maksimum hız (20,800 xg) 10 dakika 4 ° C santrifüjleyin, süpernatantı atmak, 10 dakika süreyle% 70 etanol yıkama ve kuru hava DNA pelet tekrarlayın. % 70 etanol yıkama tekrarlayın. </li><li> 25 mcL TE (10 mM EDTA) yeniden süspanse DNA, 65 yaşında sıcak ° C 5 dakika boyunca birden fazla tüpler varsa bir tüp içine birleştirmek ve TE 500 mcL hacim makyaj. </li><li> Ekle 500 mcL buz (veya 4 ° C)% 20 polietilen glikol (PEG) (1.2 M NaCl hazırlanan). Pelet DNA 4, 10 dakika süreyle maksimum hızda 10 dakika ve santrifüj buz üzerinde inkübe inversiyon ° C ile hafifçe karıştırın. Süpernatantı çıkartın ve atın ve 1 ml soğutulmuş% 70 etanol, 10 dakika süreyle kurumaya ile pelet DNA yıkayın ve 30-50 mcL TE veya moleküler sınıf su tekrar süspansiyon haline getirin. 65 yaşında DNA tabanda ısınmanın kolaylaştırılması ° -80 ° C arasında 5 dakika ve Mağaza DNA </li></ol> 3. DNA Saflık, Konsantrasyon ve Boyut Belirlenmesi: <ol><li> A 260/280 ve 260/230 oranları Nanodrop 1000 spektrofotometre veya herhangi bir başka uygun enstrümantasyon kullanarak ölçerek DNA kalitesini belirlemek . </li><li> Üreticinin talimatlarına göre Quant-iT dsDNA Assay kiti kullanılarak DNA konsantrasyonu belirleyin. </li><li> Darbe alan jel elektroforezi kullanılarak DNA boyutunu belirlemek. 0.5x TBE% 1 agaroz jel hazırlayın ve aşağıdaki parametreleri CHEF Mapper XA Sistemi kullanmak; ilk geçiş süresi 0.35 saniye, 7.67 s son bir geçiş süresi ve 120 ° 6.0 V / cm bir degrade açısı doğrusal rfaktör amping. 30 dakika için 1 x SYBR Green I ile jel Leke. 700-1000 ng toplam DNA jel arasındaki yükleyin. Bkz: Şekil. 3 tam protokol yoğunlaştırılmış bir form için. </li></ol> Temsilcisi Sonuçlar: Hücre çıkarma: Sıralı mikrobiyal hücre özleri (süpernatantlar) özler bulanıklık ekstraksiyon sayısı artış olarak azaldığını göstermiştir. Bu, birbirini izleyen her hücre çıkarma, hücre sayısında bir azalma göstermektedir. Burada dikkat edilmesi gereken önemli noktalar her ek hücre çıkarma adım kirletici tanıtımı için fırsat sağlar, hücre ekstraksiyon sayısı son hücre pelletini genel mikrobiyal topluluğun temsilcisi böylece minimize olması gerektiğini. Diğer kirlenme kaynakları yeterli steril laboratuar teknikleri benimseyerek kontrol altına alındı. Örneğin, çözümler otoklavlanmış DI su hazırlanır ve steril filtre. Konteynerler, alkol ile sterilize otoklava ve UV ışığı ve ultraviyole çapraz bağlayıcı altında tedavi edildi. DNA konsantrasyonu ve kalite belirlenmesi: 1,6 A 260/280 oranı ile protokol mat örnek gram başına yaklaşık 2 mikrogram HMW DNA (35-50 kb) (Şekil 4) vermiştir ve 0,7 A 260/230 oranı (Tablo 1). A 260/230 oranının düşük olması, 16S rRNA genleri ayrı bir çalışmada 7 gözlendi PCR amplifikasyon olarak mansap moleküler tabanlı uygulama inhibisyonu gibi görünse de. EPS ve tuzları; hypersaline paspaslar DNA&#39;nın iki ana kaynakları kirlenme dikkat etmek önemlidir. Bu nedenle bu kirleticilerin izlerini aşağı akışı uygulamaları üzerindeki etkilerini azaltmak için büyük çabalara rağmen A 260/230 oranları etkileyen olabilir. DNA büyüklükleri belirlenmesi: Darbe alan jel elektroforezi Şekil 3&#39;te gösterildiği gibi bir DNA smear sonucu aralıklı yönlü anahtarları ile zonklayan akım kullanır. Protokolü, yaklaşık 35-50 kb HMW DNA vermiştir. Bazı DNA boyutu 30 kb daha küçük olsa da, fosmid klonlama ~ 40 kb DNA ekler gerektirir ve daha büyük DNA parçalarının sağlam biyosentetik yollar için daha fazla erişim sağlayan 35 kb Yukarıdaki smear parçası olması önemlidir. Çalışmalarda, 35 kb Yukarıdaki DNA smear eksize edildi ve büyük takın vektör klonlama ve diğer moleküler uygulamalar için saflaştırılmış. <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="Şekil 1"/> Şekil 1: Eleuthera, Bahamalar üzerinde bulunan Hypersaline mikrobiyal mat örnekleme sitesi (Big Pond). <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="Şekil 2"/> Şekil 2&#39;de bu çalışmada kullanılan hypersaline mikrobiyal mat bir kesit. Mikrobiyal mat Eleuthera, Bahamalar üzerinde bulunan bir hypersaline gölet elde edildi. <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="Şekil 3"/> Şekil 3 şematik mikrobiyal hücre çıkarma, hücre parçalama, ekstraksiyon, metagenomic DNA saflaştırılması ve ilgili prosedürlerin <a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">daha büyük bir resim görmek için buraya tıklayın.</a> <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="Şekil 4"/> Şekil 4 darbe alan jel elektroforezi kullanarak çıkarılan DNA Molekül ağırlığı karakterizasyonu. Lane M HMW işaretleyici, 1 ve 2 şeritli Eleuthera hypersaline paspas yukarıdaki protokolü kullanılarak çıkarılan metagenomic DNA çoğaltır. <table border="1"><tbody><tr><td> Ekstraksiyon Metodu </td><td></td><td> Konsantrasyon ng / g </td><td> A 260/280 </td><td> A 260/230 </td></tr><tr><td> PEG </td><td> Tecrübe 1 </td><td> 1806,1 </td><td> 1,64 </td><td> 0,76 </td></tr><tr><td></td><td> Tecrübe 2 </td><td> 2010,8 </td><td> 1,61 </td><td> 0,75 </td></tr></tbody></table> Tablo 1. DNA konsantrasyonu ve kalite ölçümü mikrobiyal hypersaline mat ayıklanır.

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Given that total cell removal from complex and highly diverse microbial mat samples is not practical, the primary concern is how well the extracted cells represent the overall microbial mat community. In a previous study, PCR-DGGE analysis of microbial 16S rRNA genes showed that the five cell removal steps used in this protocol extracts cells that are representative of the overall microbial mat community7. The actual number of cell extraction steps required to provide a cell pellet that is representative of th...

<table border="1"><tbody><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> ß-mercaptoethanol </td><td> Sigma-Aldrich </td><td> M3148 </td><td></td></tr><tr><td> Polietilen glikol 8000 </td><td> Promega </td><td> V3011 </td><td> 1.2 M NaCl içinde% 20 </td></tr><tr><td> Potasyum asetat </td><td> Fisher Scientific </td><td></td><td> Fisher Scientific </td></tr><tr><td> Quant-iT dsDNA Assay kiti </td><td> Invitrogen </td><td> Q33130 </td><td></td></tr><tr><td> RNaz </td><td> Epicentre </td><td> MRNA092 </td><td></td></tr><tr><td> Sodyum klorür </td><td> BDH Kimyasallar </td><td> BDH8014 </td><td> Uygun kons. </td></tr><tr><td> Sodyum dodesil Sülfat </td><td> Fisher Scientific </td><td> 03-500-509 </td><td> % 10 su </td></tr><tr><td> sodyum hexametaphosphate </td><td> Merck Kimyasallar </td><td> SX0583-3 </td><td> Su içinde% 2 </td></tr><tr><td> TBE </td><td> Fisher Scientific </td><td> BP1333-1 </td><td></td></tr><tr><td> CHEF Mapper XA Sistemi </td><td> Bio-Rad Laboratories </td><td> 170-3670 </td><td></td></tr><tr><td> NanoDrop 1000 spektrofotometre </td><td> Thermo Scientific </td><td> ND-1000 </td><td></td></tr><tr><td> Vortexer </td><td> Bilimsel Industries Inc. </td><td></td><td></td></tr><tr><td> Ultraviyole Çapraz </td><td> UVP </td><td></td><td></td></tr><tr><td> Waring blender </td><td> Waring laboratuvar </td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

Metagenomic veri Başarılı ve doğru analiz ve yorumlama, yüksek kaliteli, yüksek molekül ağırlığı (HMW) topluluk DNA verimli çıkarma bağımlı. Ancak, çevre mat örnekleri, genellikle yüksek kalite, HMW DNA yüksek konsantrasyonlarda elde etmek için zorluklar teşkil. Hypersaline mikrobiyal paspaslar, yüksek miktarda ekstraselüler polimerik maddelerin (EPS) 1. ve çıkarılan DNA alt uygulamalar inhibe edebilir tuzları içerir. Doğrudan ve sert yöntemleri genellikle refrakter örneklerinden DNA ekstraksiyonu kullanılır. Bu yöntemler, paspaslar, yapışkan bir matris, EPS direkt lizis sırasında DNA 2,3 bağlar, çünkü genellikle kullanılır . Sert ekstraksiyon yöntemlerinin bir sonucu olarak, küçük boyutları 4,5,6 parçalanmış DNA olur. DNA, bu nedenle büyük eklemek vektör klonlama için uygun olur. Bu sınırlamaları aşmak için, biz hypersaline mikrobiyal paspaslar iyi kalite ve kantite HMW DNA ayıklamak için geliştirilmiş bir yöntem rapor. Biz karıştırma ve diferansiyel santrifüj yoluyla arka plan mat matris mikrobiyal hücrelerin ayrılması içeren dolaylı bir yöntem kullandı. Ayıklanan mikrobiyal hücrelerin DNA özü ve arındırmak için mekanik ve kimyasal işlemler bir arada kullanıldı. Bizim protokol 1,6 A 260/280 oranı, HMW DNA (35-50 kb) mat örnek gram başına yaklaşık 2 mikrogram verir. Ayrıca, 16S rRNA genleri 7 amplifikasyonu protokol bulaşanların herhangi bir inhibitör etkilerini en aza indirmek veya ortadan kaldırmak mümkün olduğunu göstermektedir . Bizim sonuçlarımız Fonksiyonel metagenomic çalışmalar için mikrobiyal paspaslar HMW DNA ekstraksiyonu için uygun bir yöntem sağlar ve diğer çevre örnekleri DNA ekstraksiyonu zorlu olduğu için geçerli olabilir.

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

Biz hypersaline mikrobiyal paspaslar yüksek molekül ağırlıklı DNA ayıklamak için geliştirilmiş bir protokol sağlar. Mikrobiyal hücrelerin DNA ekstraksiyonu ve saflaştırma mat matris önce ayrılır. Bu konsantrasyonları, kalite ve DNA boyutunu artırır. Bu protokol, diğer refrakter numuneler için kullanılabilir.

Mikrobiyal Paspas Yüksek Moleküler Ağırlıklı DNA çıkarımı

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

extraction-of-high-molecular-weight-dna-from-microbial-mats

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Biology

19.5K Görüntüleme.  Arnold School of Public Health, University of South Carolina. Biz hypersaline mikrobiyal paspaslar yüksek molekül ağırlıklı DNA ayıklamak için geliştirilmiş bir protokol sağlar. Mikrobiyal hücrelerin DNA ekstraksiyonu ve saflaştırma mat matris önce ayrılır. Bu konsantrasyonları, kalite ve DNA boyutunu artırır. Bu protokol, diğer refrakter numuneler için kullanılabilir.

Video: Extraction of High Molecular Weight DNA from Microbial Mats

Biology of Microbial Communities  - Interview

Mikrobiyal Toplulukları Biyoloji - Röportaj

Biyoloji

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.

0.22 mcM Sterivex Filtreler ve Sezyum Klorür Yoğunluk Gradient Santrifüj DNA Ekstraksiyon

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.

Topraklar ve tortulları Yüksek Moleküler Ağırlık Genomik DNA çıkarımı

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.

Genetik ve epigenetik Analizleri Parafin Gömülü Malzeme DNA Ekstraksiyon

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.

Kanatlı Üretim Örneklerden Bakteriyel Topluluklar Nitel ve Nicel Değerlendirme Karma DNA Ekstraksiyon Yöntemi

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.

Musin Agaroz jel elektroforezi: Yüksek molekül ağırlıklı glikoproteinler için Western Blot


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.

RNA ve DNA Ekstraksiyon hem Formalin sabit Parafin gömülü Doku Çekirdek hazırlanması

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

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Beyaz sinekten Endosimbonların Çıkarılması İçin Yöntemler<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

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.

Yüksek İşlemli DNA Çıkarma Plakalarının Yüklenmesinde Modifiye Yöntemler Kontaminasyon Potansiyelini Azaltır

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

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.

Caenorhabditis elegans Genomik DNA'sının Küçük Ölçekli Ekstraksiyonu

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