Name: laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers
Uploaded: 2017-10-05T12:55:00
Description: Watch this Scientific Journal Video about Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers at JoVE.com

Vi takker Silke Classen fra Research Institute for økosystemet analyse og vurdering (gaiac), RWTH Aachen for hendes hjælp i etablering af gruppe-specifikke primer sæt bruges i denne protokol. Vi takker også Peter Martin og Laura Schynawa fra universitetet i Kiel for samarbejde i vand mider eksperimenter. Vi takker også tekniske lab assistenter for at holde lab kørende og forberede register over virksomheder og katalog numre. Dette arbejde blev støttet af den tyske Forskningsfonds (DFG; projekt GE 2219/3-1).

1. indsamle Macroinvertebrates fra marken og forberede prøverne for genetiske Gut indhold analyser.
<ol><li>Indsamle macroinvertebrates på hver lokalitet, sortere ud individer af consumer art af interesse i enkelt rør, og direkte fryse dem i flydende nitrogen eller tøris til at forhindre gut clearance og nedbrydning af DNA. 
	Bemærk: Undgå at gemme prøver i alkohol, fordi nogle macroinvertebrates har tendens til at gylpe før alkohol dræber dem.</li>
	<li>Kun bruge mave-og tarmkanal af mellemstore...

<ol>
	<li>Carreon-Martinez, L., Heath, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revolution+in+food+web+analysis+and+trophic+ecology:+diet+analysis+by+DNA+and+stable+isotope+analysis.">Revolution in food web analysis and trophic ecology: diet analysis by DNA and stable isotope analysis.</a> Mol Ecol. 9 (1), 25-27 (2010).</li><li>Hardy, C. M., Krull, E. S., Hartley, D. M., Oliver, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+source+accounting+for+fish+using+combined+DNA+and+stable+isotope+analyses+in+a+regulated+lowland+river+weir+pool.">Carbon source accounting for fish using combined DNA and stable isotope analyses in a regulated lowland river weir pool.</a> Mol Ecol. 19 (1), 197-212 (2010).</li><li>Baxter, C. V., Fausch, K. D., Saunders, W. 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(380-381), 977-990 (2006).</li><li>Koester, M., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+intraguild+predation+of+Dikerogammarus+villosus+(Sowinsky,+1894)+at+an+invasion+front+in+the+Untere+Lorze,+Switzerland.">No evidence for intraguild predation of Dikerogammarus villosus (Sowinsky, 1894) at an invasion front in the Untere Lorze, Switzerland.</a> Aquat Invasions. 9 (4), 489-497 (2014).</li><li>Sonnenberg, R., Nolte, A. W., Tautz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+LSU+rDNA+D1-D2+sequences+for+their+use+in+species+identification.">An evaluation of LSU rDNA D1-D2 sequences for their use in species identification.</a> Front Zool. 4 (6), 6 (2007).</li><li>Koester, M., Bayer, B., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+Dikerogammarus+villosus+(Crustacea,+Gammaridae)+a+'killer+shrimp'+in+the+River+Rhine+system?.">Is Dikerogammarus villosus (Crustacea, Gammaridae) a 'killer shrimp' in the River Rhine system?.</a> Hydrobiologia. 768 (1), 299-313 (2016).</li><li>Koester, M., Cla&#223;en, S., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+group-specific+PCR+primers+for+the+identification+of+freshwater+macroinvertebrates.">Establishment of group-specific PCR primers for the identification of freshwater macroinvertebrates.</a> Conserv Genet Resour. 5 (4), 1091-1093 (2013).</li><li>Martin, P., Koester, M., Schynawa, L., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+detection+of+prey+DNA+in+Hygrobates+fluviatilis+(Hydrachnidia,+Acari):+a+new+approach+for+determining+predator-prey+relationships+in+water+mites.">First detection of prey DNA in Hygrobates fluviatilis (Hydrachnidia, Acari): a new approach for determining predator-prey relationships in water mites.</a> Exp Appl Acarol. 67 (3), 373-380 (2015).</li><li>Deagle, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+Seabird+Diet+through+Genetic+Analysis+of+Faeces:+A+Case+Study+on+Macaroni+Penguins+(Eudyptes+chrysolophus).">Studying Seabird Diet through Genetic Analysis of Faeces: A Case Study on Macaroni Penguins (Eudyptes chrysolophus).</a> PLoS One. 2 (9), e831 (2007).</li><li>Sint, D., Niederklapfer, B., Kaufmann, R., Traugott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group-Specific+Multiplex+PCR+Detection+Systems+for+the+Identification+of+Flying+Insect+Prey.">Group-Specific Multiplex PCR Detection Systems for the Identification of Flying Insect Prey.</a> PLoS One. 9 (12), e115501 (2014).</li><li>Zarzoso-Lacoste, D., Corse, E., Vidal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+PCR+detection+of+prey+in+molecular+diet+studies:+importance+of+group-specific+primer+set+selection+and+extraction+protocol+performances.">Improving PCR detection of prey in molecular diet studies: importance of group-specific primer set selection and extraction protocol performances.</a> Mol Ecol Resour. 13 (1), 117-127 (2013).</li><li>Vestheim, H., Jarman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+primers+to+enhance+PCR+amplification+of+rare+sequences+in+mixed+samples+-+a+case+study+on+prey+DNA+in+Antarctic+krill+stomachs.">Blocking primers to enhance PCR amplification of rare sequences in mixed samples - a case study on prey DNA in Antarctic krill stomachs.</a> Front Zool. 5 (1), 12 (2008).</li><li>Harper, G. 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Her præsenterer vi en let og omkostningseffektiv metode til PCR-baserede bestemmelse af rovdyr-byttedyr interaktioner fra feltprøver via gruppe-specifikke primere for rDNA regioner, som kan kombineres med andre ikke-molekylære analyser af de samme enheder. Der er imidlertid adskillige kritiske trin i protokollen. For det første er forsikre specificiteten af primere bruges afgørende. For det andet er det afgørende at undgå kontaminering og tab af tarm indholdet materiale under udarbejdelse af mave-tarmkanalen og ef...

Rovdyr-byttedyr interaktioner, der udgør flertallet af trofiske interaktioner og fødekæde dynamik, er centrale aspekter til at karakterisere strømme af stof og energi i hele fødevarer webs inden for og mellem økosystemer, som er et af de vigtigste mål for økologi 1. bestemmelse af kilden og strømmen af carbon og næringsstoffer fremme forståelsen af økologiske forbindelser mellem økosystemer2. Dog er økosystemer, som floder og deres afvandingsområder, ikke kun forbundet af strømme af organisk stof og næringsstoffer, men også af flytninger af organismer3. Således kan habitat ændringer afbryde strømmen af ressourcer, som forbinder disse systemer kraftigt ændre mad spind af begge økosystemer, ikke kun direkte, men også indirekte ved at ændre de respektive sammensætning af rovdyr-byttedyr Fællesskaber. For eksempel, har ændringer af mad webs vist sig at være knyttet til bevægelser af enkelt rovdyr arter (fx, regnbueørred)4. Sådanne ændringer truer potentielt biodiversiteten og funktion af akvatiske økosystemer. Derfor er analysere rovdyr-byttedyr interaktioner i feltet afgørende for at bestemme virkningen af menneskeskabte miljøforandringer såsom vand ledelsespraksis, på den indfødte biodiversitet af akvatiske økosystemer.
Da tracking trofiske forbindelser er vanskeligt i komplekse systemer, har flere tilgange fastslået at muliggøre vurdering af fodring interaktioner i felt5. Traditionelt, undersøgelser af fodring interaktioner i feltet er baseret på visuelle identifikation af bytte forbliver i dissekeret indvolde og kræver en omfattende viden om morfologiske bytte mangfoldighed. Visuelle gut indhold analyser har givet indsigt i ressourceudnyttelsen af flere grupper af forbrugere (f.eks. sæsonvariation i kost af hummer6 og fisk7,8 eller fodring præferencer af vandlopper). Dog den fysiske processen med fordøjelsen vanskeliggør visuelle gut indhold analyser og normalt misser bløde trehjulet bytte-organismer9. For arter fodring af flydende indtagelse eller visse hvirvelløse forbrugere, der intensivt comminute deres mad før indtagelse, ligesom amfipoder, er visuelle identifikation af byttedyr i tarm indholdet umuligt10. På grund af disse begrænsninger giver Molekylær analyse en lovende alternativ.
Molekylære analyser er nu blevet en fælles værktøj giver mulighed for hurtig og præcis bytte påvisning i tarm indholdet. Rækken af sådanne teknikker er mangfoldige: strategier baseret på monoklonale antistoffer eller polymerase kædereaktion (PCR) anvendes ofte, på grund af deres høje specificitet og sensitivitet11. Udvikling af nye monoklonale antistoffer er tid - og omkostningskrævende, derfor anvendelsen af andre molekylære teknikker er mere nyttigt når antistoffer ikke allerede findes11. En anden fælles tilgang er forstærkningen af regioner af deoxyribonukleinsyre (DNA), ligesom ribosomale ribonukleinsyre (RNA) gener til stede i de fleste arter, ved hjælp af universelle primer sætter12,13. Når du bruger denne teknik, er det ofte ikke muligt at identificere en lang række bytte organismer inden for blandet kilder af DNA14. En effektiv metode til at undgå sådan en ulempe er, at bruge gruppe-specifikke primer sætter for genetiske gut indhold analyser. Designet til at forstærke kun DNA regioner af bestemte målgrupper og udelukke alle andre arter15,16, aktiverer gruppe-specifikke primere identifikation af bytte organismer på den taksonomiske niveau af de angivne grupper uden tid - og omkostningskrævende sekundære analyser. Men ligesom alle gut indholdsanalyse, sådanne analyser giver kun et øjebliksbillede af fodring adfærd. Derfor anses kombinerer molekylære gut indhold analyser med analyser af tid-integrere naturlige sporstoffer (fx stabile isotoper) gavnlige1,2.
Her beskriver vi en detaljeret metode til PCR-baserede undersøgelser af rovdyr-byttedyr interaktioner ved hjælp af gruppespecifikke primer sæt for nuklear ribosomale DNA (rDNA) regioner skal kombineres med stabil isotop analyser af det samme prøvemateriale. Vi beskriver påvisning af enkelt bytte grupper via Agarosen gelelektroforese DNA. Derudover præsenterer vi en mulighed for videre downstream analyse af PCR produkter af sådanne gruppe-specifikke primere gældende, når en højere taksonomiske opløsning end primere specificitet er påkrævet. Fordi enkelt strandede DNA (ssDNA) fragmenter form tertiære konformationer, der bestemmes af deres primære sekvens17, medføre små variationer i fragmenter forstærkes af sådanne gruppe-specifikke primere konformationelle ændringer. Sådanne ændringer kan påvises ved enkelt streng kropsbygning polymorfi (SSCP) analyser med polyacrylamidgeler17,18, muliggør en mere præcis identifikation af bytte organismer (ned til artsniveau).
Mens Agarosen gelelektroforese er en fælles og billig værktøj til at visualisere DNA fragmenter og bestemme deres omtrentlige længde19, løsningen afhænger af mængden af DNA og farvning farvestoffet anvendes20. Normalt, visualisering er ligetil når du arbejder med ren DNA-prøver, men potentielt lave mængder af byttedyr DNA i tarm indholdet af forbrugerne kan komplicere scoring af Agarosen gel elektroforese resultater. Stadig, denne metode til påvisning er muligt at screene et lavt antal forbruger prøver fra feltet for en eller et par bytte grupper, men komplikation i den scoring gør screening af et stort antal prøver for flere bytte taxa meget tid intensiv og dermed upraktisk. En mere følsom metode til påvisning er en automatiseret analyse af fragmenter via kapillar elektroforese, som Derudover giver mulighed for bestemmelse af den nøjagtige længde af fragmenter21. Flere microsatellite baseret undersøgelser har vist, at det ved hjælp af forskellige fluorescerende farvestoffer som etiketter, er muligt at påvise og bestemme forskellige fragmenter af tilsvarende længde af automatiserede fragment analyse22,23, 24. Derfor, vi også fremlægge en detaljeret protokol for parallelle påvisning af DNA fra flere bytte grupper ved hjælp af PCR med mærket gruppespecifikke primer sæt og registrering via automatiseret fragment analyse med en automatiseret sequencer. Derudover præsenterer vi resultaterne fra et casestudie viser, at påvisning af bytte DNA via automatiseret fragment analyse er en tilgang, som også giver en relativ kvantificering af indtagne bytte.

<table>
	<tbody>
		<tr>
			<td>liquid nitrogen</td>
			<td>Any</td>
			<td>NA</td>
			<td>For transport of field samples of macroinvertebrates to the laboratory.</td>
		</tr>
		<tr>
			<td>1.5 ml reaction tubes - Safelock</td>
			<td>Any</td>
			<td>NA</td>
			<td>For collection of consumer specimens in the field and subsequent conservation in liquid nitrogen reaction tubes do not necessarily need to be PCR clean. To avoid exploding of the tubes, the lid of the tubes should be punctured with a needle.</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Any</td>
			<td>NA</td>
			<td>Make sure magnification is suitable to prepare the gastro-intestinaltract of the size of consumer of interest.</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Any</td>
			<td>NA</td>
			<td>To dissect the gastro-intestinal tract of consumer specimens under the stereomikroscope.</td>
		</tr>
		<tr>
			<td>Stainless steel foceps: Dumont forceps No. 7</td>
			<td>Bioform</td>
			<td>B40d</td>
			<td>This curved stainless steel forceps can sometimes be very helful to hold specimen fixed inbetween the curved part without puncturing the gastro-intestinal tract.</td>
		</tr>
		<tr>
			<td>Stainless steel foceps: Dumont forceps Electronic No. 5</td>
			<td>Bioform</td>
			<td>B40b</td>
			<td>This fine stainless steel forceps is ideally suited for the preparation of the gastro-intestinal tract of consumer specimens.</td>
		</tr>
		<tr>
			<td>DNA-ExitusPlus</td>
			<td>AppliChem</td>
			<td>A7409,1000RF</td>
			<td>Decontamination solution for the removal of DNA and RNA contaminations</td>
		</tr>
		<tr>
			<td>fuzz-free paper towel</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab scale</td>
			<td>Any</td>
			<td>NA</td>
			<td>Precision needs to be at least 0.01 g.</td>
		</tr>
		<tr>
			<td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &#956;L)</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 2.0 ml</td>
			<td>VWR</td>
			<td>211-2165</td>
			<td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 1.5 ml</td>
			<td>VWR</td>
			<td>211-2164</td>
			<td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td>
		</tr>
		<tr>
			<td>Sodium chloride &#8805;99.5%, p.a., ACS, ISO</td>
			<td>carlroth</td>
			<td>3957.3</td>
			<td>For salt extraction buffer (SEB) and 5M NaCl-solution required for extraction of DNA.</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)-aminomethane Tris</td>
			<td>carlroth</td>
			<td>4855.2</td>
			<td>For salt extraction buffer (SEB) and TBE buffer</td>
		</tr>
		<tr>
			<td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &#956;L)</td>
			<td>carlroth</td>
			<td>8043.1</td>
			<td>For salt extraction buffer (SEB) and TBE buffer</td>
		</tr>
		<tr>
			<td>Boric acid &#8805;99.8 %, p.a., ACS, ISO</td>
			<td>carlroth</td>
			<td>6943.2</td>
			<td>For TBE buffer. Not needed if commercialy available TBE buffer is obtained.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>carlroth</td>
			<td>4360.1</td>
			<td>For extraction of DNA</td>
		</tr>
		<tr>
			<td>Proteinase K lyophil.</td>
			<td>carlroth</td>
			<td>7528.1</td>
			<td>Prepare a working solution containing 10 mg/ml.</td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Quiagen</td>
			<td>85300</td>
			<td>Bead mill for homogenization of the samples.</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads, 5 mm</td>
			<td>Quiagen</td>
			<td>69989</td>
			<td>For homogenization of samples.</td>
		</tr>
		<tr>
			<td>Heating Thermoshaker</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge: himac CT 15 RE</td>
			<td>Hitachi</td>
			<td>NA</td>
			<td>Refrigerated centrifuge used for centrifugation during the extraction of DNA (Protocol 1).</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>carlroth</td>
			<td>6752.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99.8 %, p.a.</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Molecular biology grade (ddH2O)</td>
			<td>AppliChem</td>
			<td>A7398,1000</td>
			<td>nuclease-free (stream sterilized and DEPC treated) water used for re-suspension of DNA and whenever refered to ddH2O within the manuscript</td>
		</tr>
		<tr>
			<td>Unmodified Olegonucleotides</td>
			<td>Eurofins MWG Operon</td>
			<td>NA</td>
			<td>Primers unlabeled</td>
		</tr>
		<tr>
			<td>Modified olegonucleotides</td>
			<td>Metabion International AG</td>
			<td>NA</td>
			<td>Primers labelled</td>
		</tr>
		<tr>
			<td>peqGOLD Taq all inclusive</td>
			<td>VWR Peqlab</td>
			<td>01-1000</td>
			<td>Taq all inclusive for PCR reaction mixtures (reaction buffer Y and S, dNTPs and MgCl2)</td>
		</tr>
		<tr>
			<td>Primus 96 advanced gradient thermocycler or peqStar96x Universal Gradient</td>
			<td>Peqlab</td>
			<td>NA</td>
			<td>Primer sets were originally established on a Primus 96 advanced gradient thermocycler. As this cycler was quite old, we than checked if our primer sets are efficient and specific under the same conditions using the peqSta96x (which also could simulate the older model). However, tests showed that our primers work well and are still specific (see Koester et al. 2013) without using the simulation of the old machine.</td>
		</tr>
		<tr>
			<td>Standard 96 Well PCR Plates</td>
			<td>VWR Peqlab</td>
			<td>732-2879</td>
			<td>Used for PCR reactions.</td>
		</tr>
		<tr>
			<td>Low Profile Tube Stripes 0.15 ml with Flat Cap Stripes</td>
			<td>VWR Peqlab</td>
			<td>732-3223</td>
			<td>Cap stripes used to close the PCR plates.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Peqlab</td>
			<td>35-1020</td>
			<td>Used for gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical flask</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring cylinder</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal electrophoresis unit and respective combs with teeth</td>
			<td>Any</td>
			<td>NA</td>
			<td>For agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply PS3002</td>
			<td>Life Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 1 %</td>
			<td>carlroth</td>
			<td>2218.1</td>
			<td>Used to prepare staining bath for agarose gels.</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>carlroth</td>
			<td>6749.1</td>
			<td>Needded for the denaturation buffer for SSCP analyses.</td>
		</tr>
		<tr>
			<td>Bromphenol blue</td>
			<td>AppliChem</td>
			<td>A3640.0010</td>
			<td>Needed for the loading dye for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>carlroth</td>
			<td>4621.1</td>
			<td>Needed for the loading dye for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>100 bp-DNA-Ladder extended</td>
			<td>carlroth</td>
			<td>T835.1</td>
			<td>As size standard for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>UV-Documentation system</td>
			<td>Any</td>
			<td>NA</td>
			<td>To visualize gel electrophoresis results.</td>
		</tr>
		<tr>
			<td>Rotiphorese NF-Acrylamide/Bis-solution 40 % (29:1)</td>
			<td>carlroth</td>
			<td>A121.1</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Ammonium peroxydisulphate</td>
			<td>carlroth</td>
			<td>9592.2</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>foldback clips (32 mm)</td>
			<td>Any</td>
			<td>NA</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>square-shaped plastic box (21 &#215; 6.5 &#215; 5 cm)</td>
			<td>Any</td>
			<td>NA</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>carlroth</td>
			<td>23.67.1</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>RC 10 Digital chiller</td>
			<td>VWR</td>
			<td>462-0138</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Vertical electrophoresis unit P10DS (OWL Separation Systems)</td>
			<td>VWR</td>
			<td>700-2361</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Notched glass plate</td>
			<td>VWR</td>
			<td>730-0261</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Blank glass plate</td>
			<td>VWR</td>
			<td>730-0260</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Spacers</td>
			<td>VWR</td>
			<td>730-0262</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Comb with 25 teeth</td>
			<td>VWR</td>
			<td>730-0294</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Shaking platform</td>
			<td>Any</td>
			<td>NA</td>
			<td>For constant shaking of the staining bath.</td>
		</tr>
		<tr>
			<td>GelStar Nucleic Acid Stain (Lonza Rockland, Inc.)</td>
			<td>Biozym</td>
			<td>850535 (50535)</td>
			<td>For staining of polyacrylamide gels.</td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit 400bp</td>
			<td>AB Sciex</td>
			<td>608098</td>
			<td>For automated fragment analyses. Size stanard for Batch B.</td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit 600bp</td>
			<td>AB Sciex</td>
			<td>608095</td>
			<td>For automated fragment analyses. Size stanard for Batch A.</td>
		</tr>
		<tr>
			<td>Sample Loading Solution (SLS)</td>
			<td>AB Sciex</td>
			<td>608082</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Sequenzing plate - 96 Well Polypropylene PCR Microplate</td>
			<td>Costar</td>
			<td>6551</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Plate centrifuge, PerfectSpin P</td>
			<td>VWR Peqlab</td>
			<td>NA</td>
			<td>mini plate centrifuge used to spin down PCR products.</td>
		</tr>
		<tr>
			<td>Buffer plate - 96 Well EIA/RIA Clear Flat Bottom Polystyrene Not Treated Microplate</td>
			<td>Corning</td>
			<td>9017</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>GenomeLab Seperation buffer</td>
			<td>AB Sciex</td>
			<td>608012</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Separation Gel - LPAI</td>
			<td>AB Sciex</td>
			<td>608010</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Sequenzer - CEQ8000 Genetic Analysis System</td>
			<td>Beckman Coulter</td>
			<td>NA</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>GeneMarker V1.95</td>
			<td>Softgenetics</td>
			<td>NA</td>
			<td>To analyze results of automated fragment analyses.</td>
		</tr>
	</tbody>
</table>

laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers

Analysere mad webs er afgørende for en bedre forståelse af økosystemerne. For eksempel kan mad web interaktioner gennemgå alvorlige ændringer forårsaget af invasionen af fremmede arter. En nøjagtig identifikation af feltet rovdyr-byttedyr interaktioner er imidlertid vanskeligt i mange tilfælde. Disse analyser er ofte baseret på en visuel vurdering af gut indhold eller analyse af stabile isotop nøgletal (δ15N og δ13C). Sådanne metoder kræver omfattende viden omkring, henholdsvis, morfologiske forskellighed eller isotopiske signatur fra individuelle bytte organismer, fører til hindringer i den nøjagtige identifikation af bytte organismer. Visuelle gut indhold analyser undervurdere især bløde trehjulet bytte organismer, fordi maceration, indtagelse og fordøjelse af bytte organismer gør identifikation af specifikke arter vanskeligt. Dermed, polymerase kædereaktion (PCR) baseret strategier, for eksempel brugen af gruppespecifikke primer sæt, giver et stærkt værktøj til undersøgelse i food web interaktioner. Her, beskriver vi detaljerede protokoller for at undersøge gut indholdet af macroinvertebrate forbrugere fra feltet gruppe-specifikke primer sæt for nuklear ribosomale deoxyribonukleinsyre (rDNA). DNA kan være udvundet enten fra hele enheder (for lille taxa) eller tarm indholdet af prøver indsamlet i feltet. Tilstedeværelse og funktionelle effektivitet af DNA skabeloner skal bekræftes direkte fra den testede enkelte ved hjælp af universelle primer angiver målretning de respektive underenhed af DNA. Vi viser også, at forbruges bytte kan bestemmes yderligere ned til artsniveau via PCR med umodificerede gruppe-specifikke primere kombineret med efterfølgende enkelt streng kropsbygning polymorfi (SSCP) analyser ved hjælp af polyacrylamidgeler. Derudover viser vi, at anvendelsen af forskellige fluorescerende farvestoffer som etiketter muliggør parallel screening for DNA fragmenter af forskellige bytte grupper fra flere gut indhold prøver via automatiseret fragment analyse.

Vi har med held brugt trin, der beskrives i denne protokol for kost analyser inden for forskellige undersøgelser. I dette afsnit præsenterer vi eksempler der beskriver anvendelsen af de forskellige dele af protokollen.
Som et eksempel, for at demonstrere effektiviteten af gruppespecifikke primer sæt etableret af forfatterne28 og mulighederne for videre downstream analyse af PCR-produkter af SSCP analys...

Watch this Scientific Journal Video about Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers at JoVE.com

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Mest almindelige gut indhold analyser af macroinvertebrates er visuel. Der kræver intens viden om morfologiske diversitet af organismer, byttedyr, de savner bløde trehjulet bytte og, på grund af stærk comminution af byttedyr, er næsten umuligt for visse organismer, herunder amfipoder. Vi leverer detaljerede, Roman genetiske metoder til macroinvertebrate bytte identifikation i kosten af amfipoder.

Laboratoriet protokol for genetiske Gut indhold analyser af akvatiske Macroinvertebrates brug af gruppe-specifikke rDNA primere

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

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