The authors would like to acknowledge Latoya Wiggins and Katelyn Griffin for their assistance in sample acquisition, as well as Laura Lee Rutherford for their assistance in sampling and molecular analyses. We would also like to thank Sarah Owens from Argonne National Lab for microbiomic sample preparation and sequencing. These investigations were supported equally by the Agricultural Research Service, USDA CRIS Projects &#8220;Pathogen Reduction and Processing Parameters in Poultry Processing Systems&#8221; #6612-41420-017-00 and &#8220;Molecular Approaches for the Characterization of Foodborne Pathogens in Poultry&#8221; #6612-32000-059-00.

1. Mechanische Homogenisierung von Umweltzucht Proben <ol style="margin-left: 40px;"><li> Vor der Extraktion, setzen Sie ein Wasserbad auf 95 ° C und das Wasser Badezeit, um diese Temperatur zu erreichen. </li><li> Abwiegen 0,33 g Boden oder Fäkalien in eine 2 ml Lysismatrix E Rohr. <ol><li> Sie 0,33 g der Probe in der Röhre nicht überschreiten, da dies die folgenden Lösungen führen, um die Kapazität des Rohres übertrifft. </li><li> Eingefrorene Proben auf Raumtemperatur vor dem Wiegen. </li><li> Um insgesamt 1 g Boden / Kot zu analysieren, aus 3 wiegen replizieren 0,33 g Proben für jedes einzelne Umweltprobe. </li><li> Proben bei -20 ° C innerhalb der konischen Matrixröhren vor der Extraktion, wenn nötig. </li></ol></li><li> Fügen Sie 825 ul Natriumphosphat-Puffer und 275 ul des PLS-Lösung zu einem Probenröhrchen. Mischen auf dem Vortex ~ 15 Sekunden und dann Zentrifugieren Sie die Proben bei 14.000 xg für 5 min. </li><li> Dekantiert man die supernatant und fügen Sie 700 ul Puffer ASL. Mischen mit einem Vortex für 5 Sekunden. <ol><li> Sorgen Sie für eine Headspace (~ 10% Volumen) im konischen Rohr an dieser Stelle. Wenn kein Kopfraum, werden die Rohre eine Tendenz, bei der nächsten Stufe der Homogenisierung, die um eine Kreuzkontamination und / oder Probenverlust führen könnte auslaufen müssen. </li></ol></li><li> Die Proben werden in einen FastPrep 24 Instrument und Homogenisierung der Proben mit einer Geschwindigkeit von 6,0 m / s für 40 sec. </li><li> Zentrifugieren Sie die homogenisierte Probe bei 14.000 xg für 5 min. Den Überstand in ein steriles 2 ml Mikrozentrifugenröhrchen. </li><li> Um die DNA-Gewinnung aus der Probe zu maximieren, wiederholen Sie die Schritte 1.4 bis 1.6, die Kombination der Überstände in den gleichen sterilen 2 ml Mikrozentrifugenröhrchen. </li></ol> 2. Die enzymatische Hemmung der Inhibitoren von Proben Homogenate HINWEIS: Dieses Protokoll verwendet die QIAamp DNA Stool Mini Kit. <ol style="margin-left: 40px;"><li> Inkubieren Sie dieÜberstand in einem 95 ° C Wasserbad für 5 min zur DNA-Gewinnung aus verbleibenden Zellen in dem Überstand zu maximieren. <ol><li> Inkubiere bei 70 ° C für Proben, die hauptsächlich aus Gram-negative Organismen. Jedoch, wenn Gram-positive Organismen vorhanden ist (was der Fall mit Geflügel Kotproben ist) sind, Inkubation bei 95 ° C. </li><li> Verwenden Sie Kunststoff-Befestigungsklammern auf den Mikrozentrifugenröhrchen, um sicherzustellen, dass die Rohre nicht &quot;Pop&quot; offen und möglicherweise verlieren Probenvolumen als Druck kann in diesen verschlossenen Reaktionsgefäße zu bauen. </li></ol></li><li> Öffnen Sie jedes Mikrozentrifugenröhrchen, um die Druck, re-cap die Reaktionsgefäße zu lösen und mischen mit einem Vortex für 15 Sekunden. </li><li> Zentrifugieren Sie die Probe bei 14.000 · g für 1 min, entfernen Sie 1,2 ml des Überstandes und legen Sie sie in eine neue sterile 2 ml Mikrozentrifugenröhrchen. </li><li> 1 Inhibitex Registerkarte zu jeder Probe und mischen mit einem Vortex, bis die Probe wird eine gleichmäßig weiße / Aus-weiße Flüssigkeit. <ol><li> Vermeiden touching die Registerkarte Inhibitex während des Einsetzens in das Reaktionsgefäß mit der Probe. Um dies zu erreichen, setzen Sie die Blisterpackung auf die Registerkarte, die direkt über dem offenen Mikrozentrifugenröhrchen und drücken Sie auf die Registerkarte vorsichtig aus dem Blister und in das Reaktionsgefäß. </li></ol></li><li> Inkubieren der Probe für 1 min bei RT (ca. 25 ° C) und Zentrifugation bei 14.000 × g für 5 min. </li><li> Übertragen Sie die gesamte Flüssigkeit in ein steriles 1,5 ml Mikrozentrifugenröhrchen und Zentrifuge bei 14.000 xg für 5 min. <ol><li> Vermeiden jegliche verbleibenden Partikel, die am Boden des Mikrozentrifugenröhrchens am Ende von Schritt 2.5 beim Übertragen der Flüssigkeit sedimentiert haben kann. </li></ol></li></ol> 3. Automatisierte DNA-Reinigung mit dem QIAcube Robotic Workstation HINWEIS: Die Anzahl von Kunststoffzusätze, die Anordnung der Probenrotor Adapter innerhalb der Zentrifuge, und die erforderlichen Mengen der Puffer / Lösungen abhängig numzahl der Proben, die ausgeführt wird sind. <ol style="margin-left: 40px;"><li> Fügen Elutionsgefässe und Filterrohre in die entsprechenden Steckplätze innerhalb der Rotoradaptern. Für jede Probe wird mit 400 &amp; mgr; l zu dem mittleren Schlitz der Rotorteil. Zeigen die Rotoradaptern in der Workstation Zentrifuge in der richtigen Anordnung gemäß der Anzahl von Proben gereinigt wird. <ol><li> Stellen Sie sicher, dass alle der Mikrogefäßdeckel ordnungsgemäß innerhalb der Rotorteil gesichert, da ein Ausfall kann es zu in Scheren während einer der Zentrifugationsschritte des Reinigungsprotokoll führen. </li></ol></li><li> Fügen Sie die erforderliche Anzahl von 1.000 &amp; mgr; l und 200 &amp; mgr; Filter-Spitzen auf der Arbeitsstation, und füllen Sie die mitgelieferten Pufferflaschen mit dem erforderlichen Volumen von Puffer. HINWEIS: Die für dieses Reinigungsprotokoll (AL, AW1, AW2 und AE) benötigten Puffer sind alle innerhalb des QIAamp DNA Stool Mini Kit enthalten. Der Benutzer braucht, um die 100% Ethanol, die für die AW bu benötigt wird liefernob bietet und als Lösung in dem Reinigungsverfahren verwendet wird. </li><li> Fügen Sie das erforderliche Volumen der zugeführten Proteinase K-Lösung in ein steriles 1,5 ml Mikrozentrifugenröhrchen und legen Sie sie in Schlitz A auf der Arbeitsstation. Außerdem fügen die erforderliche Anzahl (gleich der Anzahl von Proben gereinigt wird) mit dem Rüttler Abschnitt der Arbeitsstelle 2 ml Safe-Lock-Mikroprobenröhrchen RB. <ol><li> Stellen Sie sicher, dass die Deckel der Probenröhrchen fest in die entsprechenden Schlitze auf der Arbeitsstation platziert, da ein Versagen zu tun, wird zu einem Fehler, wenn die Maschine zunächst scannt die Arbeitsstation, um sicherzustellen, dass alle benötigten Kunststoffe und Flüssigkeiten sind für den angeforderten verfügbar laufen. </li></ol></li><li> Verwendung des Touchscreens auf der Arbeitsstation, wählen Sie die DNA Stool - Human Hocker - Nachweis von Krankheitserregern auf Protokoll, und lesen Sie sich die folgenden Parameter, um sicherzustellen, dass die Arbeitsstation richtig eingelegt wurde. Sobald Sie alle Kontrollbildschirme übergeben, wählen Sie Start, um dieses Protokoll ausführen. <ol><li> Wenn Extrahieren von DNA aus mehr als 12 Proben, beginnen die Homogenisierung (Stufe 1) für den nächsten Satz von Proben, da ein Durchlauf von 12 Proben nimmt ~ 72 min auf der Workstation zu beenden. </li></ol></li><li> Entfernen Sie die Proben aus den Rotor-Adapter, diese abschließen, und statt bei -20 ° C bis zur anschließenden Downstream-Analysen benötigt. <ol><li> Zu diesem Zeitpunkt verbinden die 3 replicate Reinigungen für eine individuelle Probe (insgesamt analysierten Betrag = 1 g) unter Verwendung eines Zentrifugations / Verdampfungs-basierten System. Vereinige die Replikate und Wieder Eluats auf ein Endvolumen von 100 &amp; mgr; l Tris-EDTA-Puffer. </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

Die verwendeten DNA-Extraktionsverfahren erfolgen die quantitativen und qualitativen Gesamtkeim Schätzungen Gemeinschaft sowohl für die fäkale und Wurf-Proben, die Unterstützung der Probenanalysen Abhängigkeit der DNA-Extraktionsverfahren zuvor 1,3,6 gesehen. Sowohl für die fäkale und Wurf Proben war die Reihenfolge der Durchführung der DNA-Extraktionsmethoden verschiedene für die quantitative (Mechanisch&gt; Trekking&gt; enzymatische) und qualitativen (enzymatische&gt; Trekking&gt; Mechanische) Gesa...

When analyzing complex clinical or environmental samples (e.g., feces, soils), there are two main methodologies used for the extraction of DNA. The first is a mechanical disruption of the matrix using an intense bead-beating step, while the second is an enzymatic disruption of the matrix to chemically release bacterial cells and inhibit PCR inhibitors from the matrix simultaneously. Given the different means by which these two types of extraction methods work, it is not surprising that previous studies demonstrated that the appropriate DNA extraction method is both highly sample and analysis dependent. Comparative DNA extraction studies previously showed that some methods are more appropriate for improved DNA quality and quantity from environmental samples1-3, while others demonstrated improvements for community-level analyses such as denaturing gradient gel electrophoresis (DGGE)4-6, terminal restriction fragment length polymorphism (T-RFLP)7, automated ribosomal intergenic spacer analysis (ARISA)8, and phylogenetic microarrays9. Therefore, appropriate DNA extraction methods need to be used, or developed, according to the types of environmental samples and the types of analyses being performed on those samples, especially given the recent advancements in bacterial community analyses. 
Next generation sequencing, in conjunction with more quantitative community assessments (e.g., quantitative PCR (qPCR)), is becoming more prevalent in the environmental and clinical sciences, however, very little research has been performed to determine the effect of DNA extraction methods on these data sets. Most DNA extraction comparison studies dealt with microbiomic community estimates from human or human model samples10,11, not agricultural animal samples. The few poultry-focused next generation sequencing studies dealt with specific metagenomic12,13 or microbiomic14 questions; they did not discuss the effect of DNA extraction method on the resulting microbiomic analyses. Considering the complex nature of environmental samples related to poultry production (e.g., feces, litter/bedding, pasture soil), DNA extraction methods need to be carefully selected. Poultry-related environmental samples are known to contain large numbers of PCR inhibitors and up to 500-fold DNA extract dilutions have been required for PCR and subsequent downstream analysis15-17. Therefore it is essential that DNA extraction methods be optimized for these types of samples in order to not only physically disrupt the matrix, but also to be able to reduce/eliminate the large number of inhibitors that are present. 
The QIAamp DNA Stool Mini Kit, an enzymatic extraction method, has been considered the &#8220;gold standard&#8221; when extracting DNA from difficult gut/fecal samples1,18,19 and has been applied successfully to poultry environmental samples8,14. The enzymatic removal of PCR inhibitors through the use of a proprietary matrix is one of the greatest advantages of using this method for these types of environmental samples, as is the ability to significantly improve throughput (and reduce sample processing error) using automated workstations. One major disadvantage is the lack of a mechanical homogenization step to physically disassociate bacterial cells from the environmental matrix. When testing gut and fecal samples of non-poultry origin, the addition of a bead-beating or mechanical disruption step within a DNA extraction protocol significantly increased extraction efficiency9, DNA yield/quality1,4,5 and significantly improved downstream community analyses in terms of richness, diversity, and coverage5,6,11. These studies compared not only mechanical bead-beating methods to the &#8220;gold standard&#8221; enzymatic method, but some also added the mechanical bead-beating step to the enzymatic protocol to improve results6,9,11. 
According to the results from the above studies, bacterial community analyses (both qualitative and quantitative) could be improved from poultry-related environmental samples through the addition of a mechanical homogenization step to the enzymatic method. Therefore, the goal of this study was twofold: (1) to develop a novel DNA extraction technique that utilizes the most desirable aspects of both the mechanical (powerful homogenization step) and enzymatic (PCR inhibitor removal and automation) extraction methods and (2) compare the quantitative (via qPCR) and qualitative (via microbiomics) bacterial community assessments of this novel method to representative mechanical and enzymatic methods.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysing Matrix E tube</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">6914-050</td>
			<td>Different sizes available and the last 3 numbers of the cat. No. indicate size (-050 = 50 tubes, -200 = 200 tubes, -1000 = 1000 tubes)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Phosphate Solution</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">6570-205</td>
			<td>Can be purchased individually, or also contained within the FastDNA Spin Kit for Feces (Cat. No. 116570200)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PLS Buffer</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">6570-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buffer ASL (560 ml)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">19082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FastPrep 24 homogenizer</td>
			<td style="width:71px;">MPBio</td>
			<td align="right" style="width:119px;">116004500</td>
			<td>48 x 2 ml HiPrep adapter (Cat. No. 116002527) available to double throughput of mechanical homogenization step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA Stool Mini Kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">51504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAcube24 (110V)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">9001292</td>
			<td>Preliminary results show that QIAcube HT (Cat. No. 9001793) can be used to improve throughput, but different consumables are required of this machine and more comparative work needs to be done.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter-Tips, 1000 ml (1024)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter-Tips, 200 ml (1024)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990332</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAcube Rotor Adapters (10 x 24)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990394</td>
			<td>For 1.5 ml microcentrifuge tubes included with in the rotor adapter kit there is an alternative.&#160; It is Sarstedt Micro tube 1.5 ml Safety Cap, Cat. No. 72.690</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sample Tubes RB (2 ml)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990381</td>
			<td>Alternative: Eppendorf Safe-Lok micro test tube, Cat. No. 022363352</td>
		</tr>
	</tbody>
</table>

a hybrid dna extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples

Die Wirksamkeit von DNA-Extraktionsprotokollen kann stark abhängig sowohl von der Art der Probe untersucht, und die Arten von Downstream-Analysen durchgeführt werden. In Anbetracht, dass der Einsatz neuer Bakteriengemeinschaft Analysetechniken (zB microbiomics, Metagenomik) wird immer häufiger in den Agrar- und Umweltwissenschaften, und viele Umweltproben in diesen Disziplinen können physikalisch-chemisch und mikrobiologisch einzigartige (zB Fäkalien und Abfall / Bettwäsche Proben aus sein die Geflügelproduktion Spektrum), müssen geeignete und wirksame DNA-Extraktionsmethoden sorgfältig ausgewählt werden. Daher wurde eine neue halbautomatische Hybrid-DNA-Extraktionsverfahren, die speziell für die Verwendung mit Umweltgeflügelproduktion Proben entwickelt. Diese Methode ist eine Kombination der beiden wichtigsten Arten von DNA-Extraktion: mechanische und enzymatische. Ein zweistufiges intensiven mechanischen Homogenisierungsschritt (mit Wulst schlag speziell für environmen formuliertental Proben) wurde Anfang der &quot;Goldstandard&quot; enzymatische DNA-Extraktions-Verfahren hinzugefügt für Stuhlproben, um die Entfernung von Bakterien und DNA aus der Probenmatrix und zur Verbesserung der Rückgewinnung von Gram-positiven bakteriellen Mitgliedern der Gemeinschaft. Sobald die enzymatische Extraktion Abschnitt des Hybridverfahren eingeleitet wurde, wurde die restliche Reinigungsverfahren unter Verwendung eines Roboter-Arbeitsstation, um den Probendurchsatz zu erhöhen und Probenbearbeitungsfehler automatisiert. Im Vergleich zu den strengen mechanischen und enzymatischen DNA-Extraktionsmethoden, dieses neue Hybridverfahren vorgesehen die beste Gesamtleistung in Kombination, wenn man quantitativ (mit 16S-rRNA-qPCR) und qualitativen (mit microbiomics) Schätzungen der Gesamtbakteriengemeinschaften bei der Verarbeitung von Geflügel Kot und Einstreu Proben .

Für diese Studie wurden frisch fäkal Kot und Einstreu Proben von einem kommerziellen Broilerstall (~ 25.000 Vögel) im Südosten der USA gewonnen. Die Masthühner (Gallus gallus) waren Cobb-500 durchquert, und sie waren 59 Tage alt zum Zeitpunkt der Probenahme. Frische Fäkalien und Abfall-Proben wurden von vier verschiedenen Bereiche innerhalb des Hauses gewonnen (in der Nähe von Kühlkissen, in der Nähe der waterer / Zubringerlinien, zwischen den waterer / Speiseleitungen und in der Nähe von Abluftventil...

Watch this Scientific Journal Video about A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples at JoVE.com

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

Ein Hybrid-DNA-Extraktionsmethode für die qualitative und quantitative Bewertung der bakteriellen Gemeinschaften aus Zucht Proben

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

a-hybrid-dna-extraction-method-for-qualitative-quantitative

Research

JoVE Journal

Biology

14.4K Aufrufe.  USDA-Agricultural Research Service. 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. 

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

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Forschung

Biologie

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

Verwendung von Drehstabtest als Methode für die qualitative Analyse von Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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.

DNA-Extraktion von 0,22 pM Sterivex Filter und Cäsiumchlorid-Dichtegradientenzentrifugation

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.

Extraktion von High Molecular Weight genomischer DNA aus Böden und Sedimenten

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.

DNA-Extraktion aus in Paraffin eingebettete Material für genetische und epigenetische Analysen

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

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

Effiziente Nukleinsäure-Extraktion und 16S rRNA Gen-Sequenzierung für bakterielle Gemeinschaft Charakterisierung

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Filtration Isolierung von Nukleinsäuren: Eine einfache und schnelle DNA-Extraktionsmethode

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Ein verbesserter Schnittverfahren für widerspenstige Pflanzengewebeproben

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomischen Analyse Salmonella aus Lebensmitteln und Umweltproben

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Bewertung der Siderophorproduktion aus Pseudomonas aeruginosa-Isolaten

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification.
The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Autor im Rampenlicht: Quantifizierung von Siderophoren und Pyochelin für die Infektionskontrolle 

This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

Qualitative und quantitative Analyse der Siderophorproduktion aus Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...