This work was funded by the European Union Seventh Framework Program FP7/2007-2013 under grant agreement no. 265721. The authors thank the RIKILT Institute for Food Safety (NL) for their support in this project.

Not: Aşağıdaki protokol SPE% 50 h / h metanol ile C18 sorbent ve elüsyon ile 100 ml su numunesi üzerinde gerçekleştirilmiştir açıklanmaktadır. Zenginleştirilmiş örneği (ELISA) kiti linked immunosorbent bir enzim ile analizden önce,% 10 hac / hac metanol ulaşmak için seyreltilmiştir. 1. Reaktifleri hazırlanması <ol><li> Su örnekleri hazırlayın <ol><li> Önceki herhangi bir başka aşamasına, 0.2 um gözenek boyutlu filtreler ile, her 100 ml su örneği filtre. </li><li> Bir su hacmi içine bir referans çözeltisi uygun hacimde seyreltilerek istenen konsantrasyona sahip örnek başak. Örneğin, 300 mg / L&#39;lik bir konsantrasyonda E2 referans çözeltisinin 3,3 ul seyreltilmesiyle E2 100 ng / L su numunesi 100 ml hazırlanır. Bir örnek 10 ng / L E2 ile çivili almak için bu çözüme on kez sulandırmak. 1 ng / L E2 ile 100 ml &#39;lik bir numune elde etmek için bu ikinci bir on kat seyreltilir. </li><li> G, bir cam şişe içinde (modifiye edilmemiş veya kirpi), süzüldü örnek yerleştirinL45 konu. Aynı gün numune kullanılır. Küçük bir bölümünü alın başlangıçtaki konsantrasyon tahmin etmek ELISA ile analiz edilecek. </li></ol></li><li> Sıkı bir tüp içinde 50% v / v deiyonize (di) su içinde metanol ile elüsyon seyreltilerek 300 ul hazırlayın. </li></ol> SPE Column 2. Hazırlık <ol><li> İlk metanol 1600 ul ve ardından ters fazlı sorbent 40 mg di-su 400 ul ekleyerek oktadesil- silis sorbent partikülleri bir 20 mg / ml süspansiyon hazırlayın. Sıkıca kapağını kapatın ve bir girdap ile çalkalayın. </li><li> Sütununda taban zarı takılması <ol><li> Gözenek büyüklüğü 11 mikron ile tek Naylon membran seçin ve anti-toz dokusunun çift kat üzerine yerleştirin. 3 mm çaplı zımba ile, zarın iki küçük parça kesin. </li><li> Düz uç filtre forseps küçük membran tut ve kolonun bir tarafına yerleştirin. </li><li> Tüp düz taban konnektörü Vida ve sıkın. Membran nakış ve yerinde güvenli bir şekilde sorbent eklenebilir. Membran yerleştirildi nerede sonlarına doğru sütun işaret gövdesi üzerinde bir ok çizin. </li></ol></li><li> Paketlenmiş sorbent sütun hazırlanması <ol><li> Dibe doğru okun tutucuya sütun sabitleyin. Sütun dikey olarak mümkün olduğu ayarlanmalıdır. </li><li> Luer-Lock bağlayıcıları kullanarak sütun borusunun ucuna boş 10 ml tek kullanımlık şırınga takın. </li><li> Bir 20-200 ul ucu ile bir mikro-pipet hazırlayın 100 ul seviyesini ayarlamak. Bu pipet ucu biçimi hazırlanacak emici sütunun büyüklüğüne uyarlanır. Prosedür böyle 1 ml pipetler ile kullanılan olanlar gibi büyük pipet uçları ile daha zor olacaktır. </li><li> Bir girdap ile sorbent süspansiyon ajite ve hızla sütun üzerinde merkezde 100 ul pipetlemeyin. Enjekte ederken, aspire nazikçe tüm Pipetlenen çözüm Öte yandan şırıngayı kullanarak zarı yalak. Bu st atyaş, şırınga doldurma çözeltisi parçacıkların açık olmalıdır ve parçacık yatağı sonunda sütununda görülmektedir. </li><li> Çözeltide parçacıkların homojen süspansiyon sağlamak için bütün pipetleme adımları arasındaki stok süspansiyon çalkalayarak bu işlemi 2 kez daha tekrarlayın. Ortaya çıkan kolon sorbent 6 mg içerir. </li><li> Süspansiyon 300 ul yüklendi ve şırınga ile aspire kurutuldu edildiğinde, pozisyonda şırınga tutmak ve diğer elle forseps kullanılarak üstüne ikinci naylon membran yerleştirin. </li><li> Tüp ile ikinci konektörü aşağı Vida ve şırınga atmayın. SPE sütunu kullanıma hazırdır. </li></ol></li></ol> 3. Hazırlama Sistemi <ol><li> Luer-Lock konnektörleri kullanarak cihaz üzerinde SPE sütunu sıkın. Ok cihaz üzerinde gösterilen biri olarak aynı yöne işaret olmalıdır. </li><li> Vidalama cihaza örneğini içeren şişeyi bağlayınBunun üzerine GL45 emniyet kapağını sağladı. </li><li> &#39;Yıkama sıvısı&#39; rezervuar içinde eluent 200 ul yükleyin. </li><li> Yük &#39;Sulandırma&#39; rezervuar di-su 800 ul. </li><li> Ekstraksiyon aşamasında işlenmiş suyu toplamak için atık çıkışında bir şişe yerleştirin. Biçimi önemli değildir, ancak birim numune hacmi ile ilgili olarak yeterince büyük olması gerekir. </li><li> 1,5 ml minimum hacim kapasiteli algılayıcı çıkışın küçük bir şişe yerleştirin. Zenginleştirilmiş yıkama sıvısı ve seyreltme tamponu toplarken baloncuklar bu çıkışında oluşturacak şekilde 1 ml maksimum hacmi çok küçük olacaktır. </li><li> Basınç regülatörü elle döndürerek kapalı konumda olduğunu kontrol edin, ters saat yönünde daha fazla hareket etmesi mümkün değildir kadar. </li></ol> Prototip 4. SPE <ol><li> Arkasındaki düğmeye basarak prototip açın. </li><li> Kullanıcı arayüzü hazırlama ve programı seçerek <ol><li> Kullanıcı arabirimini açın. SeçinBilgisayarın iletişim portu hangi cihaz listeden bağlı ve düğme &#39;İleri&#39; tıklayın olduğu. </li><li> Değerleri pset 580 ve dpset 30 girin. Pompa çalışırken 580 ± 30 mbar&#39;da basınçlı sistem ve rezervuarlarda basıncı korumak için ayarlayacaktır. </li><li> Otomatik modu seçin. </li><li> Otomatik mod köşesinde, kutu &#39;yapılandırma dosyası yolu&#39; program dosyası yüklenemedi. </li></ol></li><li> Basınç regülatörü ayarlanması <ol><li> Pompayı başlatın. </li><li> Süleyman Demirel için okunan değer aşağı ama 320 mbar yakın kadar elle basınç regülatörü çevirin. </li><li> Pompayı durdurun. </li></ol></li><li> SPE işlemi başlatılıyor <ol><li> Otomatik mod köşesinde Basın &#39;başlangıç&#39;. Pompa açılacaktır ve çıkarma, ayrıştırma ve seyreltme adımlar otomatik olarak birbirini takip edecektir. 100 ml&#39;lik bir örnek için bütün işlem 50 dakika içinde gerçekleştirilir. </li><li>Süleyman Demirel değerini doğrulayın. Uygun bir akış oranını sağlanması için ekstraksiyon aşaması esnasında 350 mbar - Bu aralık 320 olmalıdır. </li><li> Analize kadar karanlıkta 4-5 ° C&#39;de küçük şişe ve mağaza kapatın. Analitin bozulmasını önlemek için aşağıdaki 30 saat içinde analiz gerçekleştirin. </li><li> İşlenmiş su imha ediniz. </li></ol></li><li> Sisteminin temizlenmesi Not: Sistem gereken her ekstraksiyon işlemi çapraz bulaşmayı önlemek için temizlenmesi sonra. <ol><li> GL 45 bir cam şişeye, metanol 70% v / v, 10 ml çözelti hazırlayın. </li><li> SPE sütunu çekin ve konnektörleri ile tüp takın. </li><li> Otomatik modda, &#39;temizlik&#39; dosyasını seçin ve aynı basınç ayarları ile başlatın. </li></ol></li></ol> ELISA ile Estradiol Konsantrasyon 5. Algılama <ol><li> Kullanılan ELISA kit ile birlikte verilen protokol içerisinde belirtilen konsantrasyonlarda kalibrasyon numuneleri hazırlayın. Tek Calibra hazırlayınmetanol,% 10 v / v ayarlanır, aynı örnek olarak matris ve bir kalibrasyon ile ayarlanır tion. </li><li> Üreticinin talimatlarına uygun olarak, levha üzerindeki kuyu numunenin gerekli miktarda koyun. Kalibrasyon numuneleri, filtre edildi, ancak metanol 10% v örnekleri SPE ile işlenir ve zenginleştirilmiş değil, değişikliğe uğratılmamış ve kirpi su numuneleri kullanarak / h. Tahlil ile ilişkili hatayı azaltmak için numune başına 3 kuyu kullanın. </li><li> Enzim ile reaktiflerin eklenmesi için kit, inkübasyon süresi, yıkama ve başka reaksiyon ile sağlanan protokol endikasyonları izleyin. </li><li> Bir plaka okuyucusu aletle kit üreticisinin talimatlarına göre her kuyuda optik sinyalin okuma ve verileri toplar. </li><li> Metanol,% 10 v zenginleştirilmiş örnekleri kalibrasyon eğrileri uygun aynı matrisin ile ilk numunedeki E2 konsantrasyonunu belirlemek, ve plaka okuyucusu aletin yazılımını kullanarak / h. </li></ol>

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The authors have nothing to disclose.

Immünolojik test kullanılarak analiz ve ardından su örneklerinin hazırlanması için yeni bir yöntem önerilmiştir. Enstrüman otomatik ve kullanıcı dostu bir şekilde katı faz ekstraksiyon gerçekleştirmenizi sağlar. Sistem üflenmeden önce su numunesi filtrasyon kritiktir. Çözelti içinde hala mevcut herhangi bir parçacık potansiyel olarak akışkan ağ tıkar ve SPE sütunu engelleyebilir. Diğer önemli bir adım SPE kolonundan hazırlanmasıdır. Sütununda parçacıkla...

Numune hazırlama herhangi bir analitik sürecinde önemli bir adımdır. Özel olarak, matriks etkileri, etkileşimleri azalmasını ve analit zenginleştirilmesi kaldırılması kesin sonuçlar elde edilir ve algılama düşük sınırları ulaşmak için gereklidir. Endokrin bozucu bileşikler (EDCs) nedeniyle canlı organizmalar üzerindeki etkisine özellikle kaygı olduğunda bile çevrede bulunan çok düşük seviyeleri. Doğal hormon 17β-östradiol Avrupa Su Çerçeve Direktifi kapsamında düzenlenen öncelikli maddeler listesine eklenecek AB su kirliliği İzleme Listesi mevcut ve eğilimli. Katı faz ekstraksiyonu (SPE), genel olarak, her iki kimyasal 1-5 (kromatografısi, kütle spektrometrisi) ile immünolojik 6-9 tespit yöntemleri ile, su içinde, küçük kirletici analizi için tatbik edilir. İkincisi immünoanalizler, formatlarda çok çeşitli mevcuttur ta özgü olarak, çevresel izleme alanındaki ilgisini kazanmıştıranalit rYer ve algılama düşük sınırlara ulaşır. 6, 7, 10, 11 Çeşitli enzim bağlı immünosorbent deneyleri (ELISA) ticari olarak temin edilebilir ve çok oyuklu bir plaka üzerinde aynı anda birden fazla numune analiz için olanak sağlar. İşlem birkaç saat sürebilir ardışık reaksiyon adımlarla oluşur. Reaksiyonun Nihai ürün kalibrasyon eğrisine göre hedef molekülün konsantrasyonunu belirlemek için, optik olarak tespit edilebilir. Klasik SPE prosedürler eluent buharlaştırma ile sorbent ön kurutma, örnek ekstraksiyon, yıkama, elüsyon ve konsantrasyonu içerir. Bu ekstrenin seyreltme için kullanılan çözücü, algılama yöntemine bağlı olarak seçilir. Immünolojik yöntemler için, güçlü organik çözücü etkilere yöntemin duyarlılığı miktarı. 12 Kurtarma ve ön konsantrasyonu performansları ek olarak, yöntem, aynı zamanda, basit olabilir ve maliyet etkin gerekmektedir. Prosedüründe otomasyonue insan ile ilgili hataları azaltmaya yardımcı olur. Daha önceki çalışmalarında 13 biz otomatik SPE için prototip tanıttı ve bizim yöntem deniz suyu örneklerinde doğal hormon 17β-östradiol analizi uygulanmıştır. Mevcut videoda biz SPE ve bağışıklık reaksiyonları ile algılama ile kendi özel uyumluluk geleneksel off-line ve on-line karşılaştırıldığında bizim yöntemin teknik avantajları vurgulamak istiyorum. Bu 17β-estradiyol tespiti için su numuneye uygulanan protokol açıklar. SPE oktadesil-silis (C18) sorbent faz ve elüsyon seyreltilmiş metanol ile yerine getirilmiştir gerçekleştirilir.

<table border="0" cellpadding="0" cellspacing="0" width="609">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Filter membrane 0.2 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">GNWP04700</td>
			<td style="width:167px;">For sample filtration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon membrane 11 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">NY1104700</td>
			<td>For SPE column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable biopsy punch 5 mm</td>
			<td style="width:107px;">Medical Budget</td>
			<td style="width:119px;">39302439</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nucleodur C18 ec&#160;</td>
			<td style="width:107px;">Macherey Nagel</td>
			<td style="width:119px;">713550.01</td>
			<td>50 &#956;m particle diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic sea water</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">SSWS500-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol standard</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;"></td>
			<td>300 ng/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol ELISA kit&#160;</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;">ADI-900-008</td>
			<td>96 wells, range 30 - 3000 ng/L</td>
		</tr>
	</tbody>
</table>

a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants

Çevre su numunelerinin katı faz ekstraksiyonu (SPE) için yeni bir yöntem önerilmiştir. Geliştirilen prototip maliyet-etkin ve kullanıcı dostu ve hızlı otomatik ve basit SPE gerçekleştirmenizi sağlar. Önceden konsantre edilmiş çözelti, düşük bir organik çözücü içeriğine sahip immunodeney ile analiz ile uyumludur. Bir yöntem olup 100 mL su örneklerinde doğal hormon 17β-estradiol ekstraksiyonu ve konsantrasyonu için önceden tarif edilmiştir. Ters faz SPE oktadesil- silis sorbent ile gerçekleştirilir ve elüsyon metanol 50% v / v, 200 ul ile yapılır. Elüent metanol miktarı azaltmak için, di-su eklenerek seyreltilmiştir. El SPE sütunu hazırladıktan sonra, genel işlem 1 saat içinde otomatik olarak gerçekleştirilir. İşlemin sonunda, östradiol konsantrasyonu, ticari bir enzim-bağlantılı bağışıklık-sorbent deneyi (ELISA) kullanılarak ölçülmüştür. 100-kat ön konsantrasyonu elde edilir ve yalnızca 10% v / v metanol içeriği. Molekülün tam geri kazanım1 ng ile elde edilir / L-de iyonize ve sentetik deniz suyu örnekleri çivili.

Sorbent ambalaj tekrarlanabilirliği kurutma ile değerlendirildi ve cam viyaller içinde pipetlendi sorbentin ağırlık ve ilk ve pre Şekil 2. Konsantrasyon gösterildiği gibi sonuç, 100 mi örnekler için test edildi enjeksiyonu sırasında Şekil 1. Tekrarlanabilirliği de gösterilmiştir edildi Konsantre örnekleri 17β-estradiol için ticari bir ELISA kiti kullanılarak tespit edilmiş ve Şekil 3&#39;te gösterilmektedir çivi...

Watch this Scientific Journal Video about A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants at JoVE.com

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Küçük Kirleticilerin İmmünolojik Analizi Su Örneklerinin Otomatik Katı Faz Ekstraksiyon Basit Bir Yöntem

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

a-simple-method-for-automated-solid-phase-extraction-water-samples

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

13.6K Görüntüleme.  Centre Suisse d'Electronique et de Microtechnique. A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Video: A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Sudan Viral Konsantrasyon için Küçük Bir Ses Prosedürü

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

Çevre

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

Enterovirus Kültür ve RT-qPCR Su Norovirüs Oluşum EPA Metot 1615 Ölçüm. Virüs Örneklerinin I. Koleksiyonu

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Chironomidae (Diptera) kullanılması Yüzey Yüzer Su Organları bir Rapid Bioassessment Protokolü Pupa Exuviae

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Otomatik Jel Boyut Seçimi Çevre Su Örneklerinden hazırlanmıştır nesil Sıralama Kütüphaneler Kalitesi Geliştirmek için

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Filtrasyon Su Örnekleri ve Çevre DNA Ekstraksiyon için bir Filtre Kartuşu Kullanımı

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Deniz Yüzey ve Numune Analizi Microplastics Örnekleme Protokolü

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Çok Örnekli Deneylerde Toprak Mikroplarını Karakterize Etmek İçin Bir Lipid Ekstraksiyon ve Analiz Metodu

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastik Peletlerden Organochlorine Pestisitlerin Çıkarılması ve Plastik Tipi Analizi

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Bir basit tekniği sık su baskını/taşkın oluştuğu için yeniden kurulması ağaç dikme

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.