Ringraziamo i membri delle Blaschke, Grunwald, e Wittwer laboratori per consulenza e assistenza tecnica. Questo lavoro è supportato dalla Fondazione PCMC, NIH R01 MH092256 e DP2 MH100008, e la March of Dimes Foundation assegno di ricerca # 1-FY13-425, a JLB.

1. Preparazione del DNA <ol><li> Preparare DNA tampone di lisi: 50 mM KCl, 10 mM Tris-HCl pH 8,3, 0,3% Tween 20, 0,3% NP40. Aggiungi fresco proteinasi K a una concentrazione finale di 1 mg / ml il giorno di utilizzo 12. </li><li> Collezione Tessuto: <ol><li> Per l&#39;adulto clip di pinna di pesce: <ol><li> Anestetizzare il pesce: Mettere il pesce in 0,004% MS-222 la soluzione (tricaine). Attendere fino a quando il movimento branchia rallenta. </li><li> Mettere pesce su una pila di 5-10 Kimwipes e tagliare un piccolo pezzo di pinna caudale, circa 2-3 mm, con una lama di rasoio sterile. </li><li> Introdurre rapidamente il pesce in un serbatoio etichettato con acqua dolce per recupero; etichettare accuratamente sia il serbatoio e tubo corrispondente che conterrà la clip pinna. Cambiare l&#39;acqua e alimentare il pesce ogni altro giorno. </li><li> Sollevare la clip pinna con un puntale sterile e trasferirlo in un tubo riempito con 100 microlitri di buffer di lisi del DNA. </li><li> Gettare la punta della pipetta, e scartare il lametta in un apposito contenitore. </li></ol></li><li> Per la clip di embrione di coda: <ol><li> Mettere gli embrioni in 0,004% MS-222 la soluzione (tricaine). Attendere 2 min. </li><li> Tagliare un pezzo di coda con una pinza sotto un microscopio da dissezione. </li><li> Mettere la coda in una provetta riempita con 30 microlitri DNA tampone di lisi. </li><li> Mettere rapidamente l&#39;embrione in un tubo contenente 100 ml paraformaldeide al 4%. </li><li> Etichettare le provette con embrioni e dei loro tubi di coda corrispondenti. </li><li> Conservare le provette con embrioni a 4 ° C durante la notte per il fissaggio. </li><li> Pulire le pinze con acqua Milli-Q e Kimwipes tra ogni embrione per minimizzare la contaminazione. </li></ol></li><li> Per gli embrioni interi: <ol><li> Mettere gli embrioni in 0,004% MS-222 la soluzione (tricaine). Attendere 2 min. </li><li> Mettere 1-5 embrioni in un tubo riempito con 100 microlitri di tampone di lisi DNA. Dechorionation non è necessario. </li></ol></li></ol></li><li> Digestione DNA Incubare le provette con il tessuto (o pinna di coda clip o embrioni) a 55 ° Cper 4 ore fino a tutta la notte 12. </li><li> Inattivare il proteinasi K Tubi di calore a 95 ° C per 15 min 12. DNA deve essere usato per la PCR immediatamente o conservato a -20 ° C per 3 mesi. </li></ol> 2. PCR <ol><li> Primer design manualmente o mediante iniettore software di progettazione (es. Lightscanner Primer Design Software-Biofire). I prodotti di PCR per HRMA sono di solito 50-200 bp; prodotti di PCR per il rilevamento SNP dovrebbero essere più piccole, tipicamente 50-80 bp. </li><li> PCR <ol><li> Eseguire reazione di PCR in una piastra a 96 pozzetti o 384 pozzetti. </li><li> Utilizza 10 microlitri volume totale: 4 microlitri master mix LightScanner (0,1 U hot-start Taq-AB polimerasi, tampone, 0.2 dNTP mm, 2 mm cloruro di magnesio, e 1x fluorescente DNA-binding dye a doppio filamento); 5 pmol ciascun primer, e 1 ml modello di DNA genomico estratto da pinna caudale adulti o 3 ml di DNA genomico da coda embrionale 13. </li><li> Campioni Coprire con 30 ml di olio minerale. </li&gt; <li> Coprire la piastra con un sigillo adesivo otticamente trasparente. </li><li> Ottimizzare PCR-ciclismo condizioni tipiche condizioni sono 95 ° C per 5 minuti e 30 cicli di 10 secondi a 95 ° C, 25 sec a 60 ° C, 30 sec a 72 ° C, che termina con 95 ° C per 30 sec e raffreddati a 15 ° C. </li><li> Conservare le piastre PCR a 4 ° C o direttamente continuano a HRMA. </li><li> Confermare il prodotto di PCR mediante analisi della sua dimensione mediante elettroforesi su gel di agarosio durante l&#39;ottimizzazione iniziale di PCR, e se indicato, mediante sequenziamento del prodotto PCR. </li></ol></li></ol> 3. HRMA <ol><li> Piastre di calore <ol><li> Posizionare la piastra in un sistema di analisi melt. </li><li> Aprire il software (LightScanner Software Call-IT 2.0). </li><li> Piastre di calore 60-95 ° C. </li><li> Creare un nuovo file di archiviazione dei dati. </li></ol></li><li> Analizzare i dati HRMA (Figura 2). Più passaggi di analisi sono possibili. Mostriamo il set più comunemente usato di dati manipulations. <ol><li> Impostazione sottoinsieme Fare clic sulla scheda sottoinsiemi. Selezionare i pozzetti con i campioni. </li><li> Normalizzazione <ol><li> Selezionare la scheda Normalize nel pannello di sinistra. Per eliminare la fluorescenza varianza, posizionare manualmente la doppia linea parallela in pre-(iniziale) e le regioni posto (finale)-fusione come illustrato (Figura 2C, punte di freccia) e normalizzare prefusione e fluorescenza post-fusione segnali di tutti i campioni a 1 e 0 rispettivamente 14. </li><li> Regolare il posizionamento delle linee in modo che la regione di fusione è nella regione tra le linee pre-e post-fusione, ma non selezionato. In generale, la Bassa Min e Max Inferiore (e Alto Min e Max Superiore) le linee di temperatura devono essere posizionati a circa 1 ° C a parte. </li><li> Scegliere posizioni di temperatura per la normalizzazione tale che tutti i campioni hanno (100%) fluorescenza massima o totale per la regione Premelt e minimo o nullo (0%) fluorescenza per la regione post-fusione (come meglio posbile). Normalizzazione dovrebbe comportare una linea quasi orizzontale nella fluorescenza massimo 1 che si estende fino al punto di fusione e poi una linea quasi orizzontale dal punto di fusione alla fluorescenza minimo di 0, non ci dovrebbe essere livelli di fluorescenza 1 o minori di 0 . Per esempio, nella Figura 2C, normalizzare le curve prefusione utilizzando intervalli di temperatura di 79-80 ° C. </li></ol></li><li> Raggruppamento / Clustering Distinguere genotipi in base alla loro temperatura di fusione (Figura 2C, scatola verde). Selezionare Raggruppamento. <ol><li> Selezionare Autogroup dall&#39;elenco di selezione Standards sotto la sezione Raggruppamento. </li><li> Selezionare Normale o Alta per la fusione dei profili con i singoli passaggi o più transizioni rispettivamente dalla lista di selezione di sensibilità nella sezione Raggruppamento. </li><li> Seleziona ComputeGruppi nella sezione Raggruppamento. </li><li> Vai al menu File e fare clic su Salva per salvare i risultati. </li></ol></li></ol></li></ol>

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PCR combinata con HRMA è una potente tecnologia per la genotipizzazione zebrafish. I vantaggi di questo approccio sono la velocità, robustezza e sensibilità per rilevare anche mutazioni puntiformi. L&#39;intero protocollo, dalla pinna-clip analisi per fondere curva, può essere eseguito in meno di otto ore da un singolo individuo. Inoltre, la tecnica è suscettibile di screening ad alto rendimento, non richiede l&#39;uso di bromuro di etidio, ed è sigillato per tutta PCR e fasi di analisi che aiuta a ridurre i probl...

Zebrafish (Danio rerio) è un sistema modello vertebrato ampiamente usato per studi di sviluppo e modellazione della malattia. Recentemente, numerose tecnologie transgeniche e mutazione sono stati sviluppati per zebrafish. Tecniche di transgenesi rapida, di solito basati su un sistema di trasposoni Tol2 1, sono stati combinati con le opzioni di clonazione migliorate per aerei frammento di DNA di montaggio 2. Nucleasi zinc-finger (ZFNs) e trascrizione nucleasi effettrici attivatore-like (Talens) sono stati utilizzati per indirizzare loci in entrambe le cellule somatiche e germinali in zebrafish 3,4. Queste tecniche possono efficacemente generare animali geneticamente modificati, con la creazione mutazione ad alta frequenza e la trasmissione germinale 3,4. Nonostante questi progressi, tecniche di genotipizzazione tradizionali in zebrafish limitano tutta la potenza degli strumenti di mutagenesi e transgenesi. PCR seguita da elettroforesi su gel, a volte combinati con RESTRIZIONn digestione enzimatica, è ampiamente utilizzato per rilevare modifiche genoma, ma richiede tempo e meno sensibile per identificare piccole inserzioni o delezioni. Saggi sonda TaqMan hanno elevati costi iniziali e richiedono un&#39;attenta ottimizzazione. Sequenziamento dei prodotti di PCR può richiede parecchi giorni e non è pratico per lo screening su larga scala. Frammento di restrizione polimorfismo della lunghezza (RFLP) analisi può discriminare solo SNPs che colpiscono un numero limitato di siti di riconoscimento degli enzimi di restrizione. Ad alta risoluzione fusione analisi (HRMA), un post-PCR metodo di analisi chiuso tubo, è un metodo sviluppato di recente che è rapida, sensibile, poco costoso, e suscettibili di screening di un gran numero di campioni. HRMA può essere utilizzato per rilevare SNP, mutazioni, e transgeni 5-7. HRMA è basato su denaturazione termica di DNA a doppio filamento, e ogni amplicone PCR ha un unico dissociazione (melt) caratteristica 5. I campioni possono essere discriminati a causa to la loro diversa composizione nucleotidica, contenuto di GC, o lunghezza, tipicamente in combinazione con un colorante fluorescente che si lega solo a doppio filamento di DNA 8. Così, HRMA può distinguere diversi genotipi in base alle diverse caratteristiche melt-curva. Poiché HRMA utilizza reagenti economici ed è un passo singolo processo di post-PCR, può essere utilizzato per strategie ad alto rendimento. HRMA è non distruttiva, così in seguito all&#39;analisi gli ampliconi della PCR possono essere utilizzati per altre applicazioni. HRMA è stato applicato in molti organismi e sistemi, inclusi linee cellulari, topi e nell&#39;uomo 9-11. Il suo uso è stato recentemente descritto in zebrafish per rilevare le mutazioni indotte da nucleasi dito di zinco (ZFNs) e Talens 6,12,13. In questo articolo, descriviamo come eseguire HRMA PCR-based in zebrafish embrionali e adulte (Figura 1). Questo protocollo è adatto per rivelare SNP, transgeni, e mutazioni, comprese SINGLE modifiche base-pair, inserimenti o eliminazioni.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">100 Reaction LightScanner Master Mix</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">HRLS-ASY-0002</td>
			<td style="width:157px;">www.biofiredx.com</td>
			<td style="width:167px;">Store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Hard-Shell PCR 96-well BLK/WHT Plates</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">HSP9665</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Microseal &#39;B&#39; Adhesive Seals</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">MSB1001</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">96-Well LightScanner Instrument</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">LSCN-ASY-0040</td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Software with Call-IT 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">High-Resolution Melting Analysis 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Primer Design Software</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vector NTI Software</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:141px;"></td>
			<td>www.invitrogen.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tricaine</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Paraformaldehyde</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid and efficient zebrafish genotyping using pcr with high-resolution melt analysis

Zebrafish è un potente sistema modello vertebrato per studiare lo sviluppo, la modellazione della malattia, e l&#39;esecuzione di screening di stupefacenti. Recentemente una varietà di strumenti genetici sono state introdotte, tra cui più strategie per indurre mutazioni e la generazione di linee transgeniche. Tuttavia, lo screening su larga scala è limitato con metodi tradizionali di genotipizzazione, che sono termini di tempo e di manodopera. Qui si descrive una tecnica per analizzare i genotipi di zebrafish per PCR combinato con alta risoluzione fusione di analisi (HRMA). Questo approccio è rapido, sensibile e poco costoso, con un minor rischio di artefatti contaminazione. Genotipizzazione mediante PCR con HRMA può essere utilizzato per gli embrioni o pesci adulti, anche nei protocolli di screening high-throughput.

Il protocollo può essere eseguita durante un singolo giorno o separato in fasi di diversi giorni (diagramma di flusso di lavoro è mostrata in Figura 1). Estrazione del DNA è seguita dalla fusione e analisi di ampliconi PCR. Le temperature per la fusione del amplicone dipendono dalle dimensioni e GC-contenuti, ma iniziano in genere e temperature finali di 50 ˚ C e 95 ˚ C siano appropriate (Figure 2A e 2B). Una volta eseguita la fusione, analisi delle curve di fluore...

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Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

PCR combinata con alta risoluzione melt analisi (HRMA) è dimostrato come un metodo rapido ed efficace al genotipo zebrafish.

Rapido ed efficiente Zebrafish genotipizzazione Uso PCR con ad alta risoluzione Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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22.1K Views.  University of Utah School of Medicine.  PCR combinata con alta risoluzione melt analisi (HRMA) è dimostrato come un metodo rapido ed efficace al genotipo zebrafish. 

Video: Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Genotipizzazione rapida del tessuto mouse utilizzando Sigma Extract-N-Amp PCR Tissue Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

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Biologia

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

Rapido PCR termici utilizzando microscala convezione termica

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Utilizzando ad alta risoluzione Tomografia Computerizzata Per visualizzare la struttura tridimensionale e funzione delle piante Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Isolamento efficiente e rapida di embrioni nelle prime fasi di<em&gt; Arabidopsis thaliana</em&gt; Semi

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Alta Risoluzione intero monte<em&gt; In Situ</em&gt; Ibridazione in embrioni di zebrafish per studiare l&#39;espressione genica e la funzione

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Analisi rapida ed esplorazione di microscopia a fluorescenza Immagini

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analisi qPCRTag - A High Throughput, Real Time PCR Assay per Sc2.0 genotipizzazione

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Ad alta risoluzione Imaging e analisi dei singoli microtubuli astrali Dynamics in germogliamento lievito

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Rapido ed efficiente Zebrafish genotipizzazione Uso PCR con ad alta risoluzione Melt Analysis

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