Name: use of a filter cartridge for filtration of water samples and extraction of environmental dna
Uploaded: 2016-11-25T13:00:00
Description: Watch this Scientific Journal Video about Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA at JoVE.com

This study was supported as basic research by CREST from the Japan Science and Technology Agency (JST) and by grants from JSPS/MEXT KAKENHI (Number 26291083) and the Canon Foundation to M.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

הערה: פרוטוקול זה אינו עוסק מי דגימה ושיטות metabarcoding. מים אפשר יהיה לטעום באופנים שונים בהתאם למטרות המחקר 16 ותראו מייה et al. 12 לפירוט אודות שיטות metabarcoding באמצעות פריימרים MiFish. ראוי לציין, כי המים שנדגמו צריכים להישמר מאוד קרים מסוננים בתוך כמה שעות, כדי ?...

<ol>
	<li>Takahara, T., Minamoto, T., Doi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+environmental+DNA+to+estimate+the+distribution+of+an+invasive+fish+species+in+ponds.">Using environmental DNA to estimate the distribution of an invasive fish species in ponds.</a> PLoS ONE. 8, e56584 (2013).</li><li>Takahara, T., Minamoto, T., Yamanaka, H., Doi, H., Kawabata, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+fish+biomass+using+environmental+DNA.">Estimation of fish biomass using environmental DNA.</a> PLoS ONE. 7, e35868 (2012).</li><li>Sigsgaard, E. E., Carl, H., M&#248;ller, P. R., Thomsen, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+near-extinct+European+weather+loach+in+Denmark+based+on+environmental+DNA+from+water+samples.">Monitoring the near-extinct European weather loach in Denmark based on environmental DNA from water samples.</a> Biol. Conserv. 183, 48-52 (2015).</li><li>Jerde, C. L., 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=Detection+of+Asian+carp+DNA+as+part+of+a+Great+Lakes+basin-wide+surveillance+program.">Detection of Asian carp DNA as part of a Great Lakes basin-wide surveillance program.</a> Can. J. Fish. Aquat. Sci. 70, 522-526 (2013).</li><li>Jerde, C. L., Mahon, A. R., Chadderton, W. L., Lodge, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&amp;#34;Sight-unseen&amp;#34;+detection+of+rare+aquatic+species+using+environmental+DNA.">&amp;#34;Sight-unseen&amp;#34; detection of rare aquatic species using environmental DNA.</a> Conserv. Lett. 4, 150-157 (2011).</li><li>Mahon, A. R., 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=Validation+of+eDNA+surveillance+sensitivity+for+detection+of+Asian+carps+in+controlled+and+field+experiments.">Validation of eDNA surveillance sensitivity for detection of Asian carps in controlled and field experiments.</a> PLoS ONE. 8, e58316 (2013).</li><li>Minamoto, T., Yamanaka, H., Takahara, T., Honjo, M. N., Kawabata, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+of+fish+species+composition+using+environmental+DNA.">Surveillance of fish species composition using environmental DNA.</a> Limnology. 13, 193-197 (2012).</li><li>Keskin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+invasive+freshwater+fish+species+using+environmental+DNA+survey.">Detection of invasive freshwater fish species using environmental DNA survey.</a> Biochem. Syst. Ecol. 56, 68-74 (2014).</li><li>Wilcox, T. M., 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=Robust+detection+of+rare+species+using+environmental+DNA:+the+importance+of+primer+specificity.">Robust detection of rare species using environmental DNA: the importance of primer specificity.</a> PLoS ONE. 8, e59520 (2013).</li><li>Thomsen, P. F., 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=Detection+of+a+diverse+marine+fish+fauna+using+environmental+DNA+from+seawater+samples.">Detection of a diverse marine fish fauna using environmental DNA from seawater samples.</a> PLoS ONE. 7, e41732 (2012).</li><li>Kelly, R. P., 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=Harnessing+DNA+to+improve+environmental+management.">Harnessing DNA to improve environmental management.</a> Science. 344, 1455-1456 (2014).</li><li>Miya, M., 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=Mifish,+a+set+of+universal+PCR+primers+for+metabarcoding+environmental+DNA+from+fishes:+detection+of+more+than+230+subtropical+marine+species.">Mifish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species.</a> Roy. Soc. Open Sci. 2, 150088 (2015).</li><li>Port, J. A., 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=Assessing+vertebrate+biodiversity+in+a+kelp+forest+ecosystem+using+environmental+DNA.">Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA.</a> Mol. Ecol. 25, 527-541 (2015).</li><li>Yamamoto, S., 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=Environmental+DNA+provides+a+'snapshot'+of+fish+distribution:+a+case+study+of+Japanese+jack+mackerel+in+Maizuru+Bay,+Sea+of+Japan.">Environmental DNA provides a 'snapshot' of fish distribution: a case study of Japanese jack mackerel in Maizuru Bay, Sea of Japan.</a> PLoS ONE. 11, e0149786 (2016).</li><li>Valentini, A., 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=Next+generation+monitoring+of+aquatic+biodiversity+using+environmental+DNA+metabarcoding.">Next generation monitoring of aquatic biodiversity using environmental DNA metabarcoding.</a> Mol. Ecol. 25, 929-942 (2016).</li><li>Rees, H. C., Maddison, B. C., Middleditch, D. J., Patmore, J. R., Gough, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+The+detection+of+aquatic+animal+species+using+environmental+DNA+-+a+review+of+eDNA+as+a+survey+tool+in+ecology.">Review: The detection of aquatic animal species using environmental DNA - a review of eDNA as a survey tool in ecology.</a> J. Appl. Ecol. 51, 1450-1459 (2014).</li><li>Stewart, F. J., DeLong, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microbial metagenomics, Metatranscriptomics, and metaprotenomics Vol. 531 Methods in Enzymology. 10, 187-218 (2013).</li><li>Walsh, D. A., Zaikova, E., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+volume+(20L+)+filtration+of+coastal+seawater+samples.">Large volume (20L+) filtration of coastal seawater samples.</a> J Vis Exp. (28), e1161 (2009).</li><li>Smalla, K., Akkermans, D. L., Elsas, J. D., Bruijn, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Microbial Ecology Manual. , 13-22 (1995).</li><li>Thomsen, P. F., Willerslev, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+DNA+-+An+emerging+tool+in+conservation+for+monitoring+past+and+present+biodiversity.">Environmental DNA - An emerging tool in conservation for monitoring past and present biodiversity.</a> Biol. Conserv. 183, 4-18 (2014).</li><li>Pedersen, M. W., 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=Ancient+and+modern+environmental+DNA.">Ancient and modern environmental DNA.</a> Phil. Trans. R. Soc. B. 370, 20130383 (2015).</li><li>Fukumoto, S., Ushimaru, A., Minamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+basin+scale+application+of+environmental+DNA+assessment+for+rare+endemic+species+and+closely+related+exotic+species+in+rivers:+a+case+study+of+giant+salamanders+in+Japan.">A basin scale application of environmental DNA assessment for rare endemic species and closely related exotic species in rivers: a case study of giant salamanders in Japan.</a> J. Appl. Ecol. 52, 358-365 (2015).</li><li>Yamanaka, H., Minamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+environmental+DNA+of+fishes+as+an+efficient+method+of+determining+habitat+connectivity.">The use of environmental DNA of fishes as an efficient method of determining habitat connectivity.</a> Ecol. Indicators. 62, 147-153 (2016).</li><li>Moss, J. A., 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=Ciliated+protists+from+the+nepheloid+layer+and+water+column+of+sites+affected+by+the+Deepwater+Horizon+oil+spill+in+the+Northeastern+Gulf+of+Mexico.">Ciliated protists from the nepheloid layer and water column of sites affected by the Deepwater Horizon oil spill in the Northeastern Gulf of Mexico.</a> Deep Sea Res. Pt I. 106, 85-96 (2015).</li><li>Hilton, J. A., Satinsky, B. M., Doherty, M., Zielinski, B., Zehr, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metatranscriptomics+of+N2-fixing+cyanobacteria+in+the+Amazon+River+plume.">Metatranscriptomics of N2-fixing cyanobacteria in the Amazon River plume.</a> The ISME journal. 9, 1557-1569 (2015).</li><li>Deiner, K., Walser, J. -. C., M&#228;chler, E., Altermatt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+capture+and+extraction+methods+affect+detection+of+freshwater+biodiversity+from+environmental+DNA.">Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA.</a> Biol. Conserv. 183, 53-63 (2015).</li><li>Eichmiller, J. J., Miller, L. M., Sorensen, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+techniques+to+capture+and+extract+environmental+DNA+for+detection+and+quantification+of+fish.">Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.</a> Mol. Ecol. Res. 16, 56-68 (2016).</li><li>Lemarchand, K., Pollet, T., Lessard, V., Badri, M. A., Micic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Sample Preparation Techniques for Soil, Plant, and Animal Samples'Springer Protocols Handbooks. , 325-339 (2016).</li><li>Turner, C. R., 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=Particle+size+distribution+and+optimal+capture+of+aqueous+macrobial+eDNA.">Particle size distribution and optimal capture of aqueous macrobial eDNA.</a> Methods Ecol. Evol. 5, 676-684 (2014).</li><li>Barnes, M. A., Turner, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+of+environmental+DNA+and+implications+for+conservation+genetics.">The ecology of environmental DNA and implications for conservation genetics.</a> Conserv. Genet. 17, 1-17 (2016).</li><li>Sorokulova, I., Olsen, E., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopolymers+for+sample+collection,+protection,+and+preservation.">Biopolymers for sample collection, protection, and preservation.</a> Appl. Microbiol. Biotechnol. 99, 5397-5406 (2015).</li><li>Renshaw, M. A., Olds, B. P., Jerde, C. L., McVeigh, M. M., Lodge, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+room+temperature+preservation+of+filtered+environmental+DNA+samples+and+assimilation+into+a+Phenol-Chloroform-Isoamyl+alcohol+DNA+extraction.">The room temperature preservation of filtered environmental DNA samples and assimilation into a Phenol-Chloroform-Isoamyl alcohol DNA extraction.</a> Mol. Ecol. Res. 2014, (2014).</li></ol>

במחקרים רבים metabarcoding באמצעות דגימות סביבתיות כגון מים ואדמה, טיפול סינון לתפקיד מחסנית המסנן הוא בדרך כלל כדלקמן 24,25: 1) חיתוך פתוח או פיצוח דיור עם כלי יד (חותך צינורות או צבת); 2) הסרת המסנן מהמחסנית; ו -3) חיתוך המסנן לחתיכות קטנות עם סכין גילוח להפקת DNA. כדי להימנע ...

DNA הסביבה (עדנה) בסביבות מימיות מתייחס לחומר גנטי המצוי בעמודת המים. מחקרים שנעשו לאחרונה הדגימו את התועלת של עדנה לאיתור דגים מסביבות מימיים שונים, כולל בריכות 1-3, נהרות 4-8, נחלים 9, ומי ים 10-14. רוב המחקרים האלה התמקדו גילוי של יחיד או כמה פולשני 1,4-6,8,14 ונדירים או מאוימים מינים 3,9, בעוד מחקרים שנעשו לאחרונה כמה ניסו זיהוי סימולטני של מינים רבים בקהילות הדגים המקומי 7,9, 12,13,15 ו mesocosms 11,12. הגישה האחרונה נקראת &quot;metabarcoding&quot; ועדנה metabarcoding משתמש וקבוצה אחת או מספר של פריימרים PCR כדי coamplify אזור הגן על פני דגימות מגוונות טקסונומית. זה ואחריו כנת ספרייה עם אינדוקס בנוסף מתאם, ואת הספריות באינדקס מנותחות על ידי רצף מקביל תפוקה גבוההפּלַטפוֹרמָה. לאחרונה מייה et al. 12 פיתח פריימרים PCR אוניברסלי עבור metabarcoding עדנה מן הדגים (המכונה &quot;MiFish&quot;). פריימרים MiFish ולמקד לאזור hypervariable של הגן המיטוכונדריה 12S rRNA (163-185 נ&quot;ב), אשר מכיל מידע מספיק כדי לזהות דגים למשפחה, הסוג ומינים טקסונומיות למעט במבניהם כמה קשורים זה לזה. עם השימוש של פריימרים אלו metabarcoding עדנה, מייה et al. 12 זוהה יותר מ -230 מינים ימיים סובטרופי מטנקים אקווריום עם שוניות אלמוגים והרכב מינים ליד האקווריום. תוך אופטימיזציה של פרוטוקול metabarcoding כדי להתאים מי ים טבעי עם רמות משתנות של ריכוז עדנה מן הדגים, יש לנו לב כי פריימרים MiFish נכשלו מדי פעם כדי להגביר את אזור היעד להכנת ספרייה שלאחר מכן. אחת הסיבות סבירות יותר עבור הגברת PCR מוצלחת זה הוא חוסר הכמות מספקת של template DNA הכלול בנפחים קטנים של מים מסוננים (כלומר 1-2 L). למרות ריכוז עדנה מקבוצת טקסונומיות ספציפית הוא לידיעה לפני ההגברה, סינון של כרכי מים גדולים (&gt; 1-2 L) תהיה דרך פשוטה ויעילה כדי לאסוף יותר עדנה מן הסביבות המימיות עם שפע דגים נדיר ביומסה, כגון מערכות אקולוגיות תחת כיפת האוקיינוס ​​במעמקי הים. ביחס מסננים סיבים דיסק כמקובל בשימוש במספר של מחקר עדנה דגי 16, מחסניות מסננות יש את היתרון של אדיב כרכי מים גדולים לפני סתימת 17. למעשה, מחקר שנערך לאחרונה הראה נפח גדול (&gt; 20 L) סינון של דגימות מי ים החוף באמצעות מחסניות מסננות 18. בנוסף, הם בנפרד ארוזים וסטרילי, וכמה מדרגות העבודה הניסיונית יכולות להתבצע בית המסנן, ובכך להקטין את ההסתברות של זיהום מהמעבדה 19. האחרוןתכונה חיונית עדנה metabarcoding, שבו את הסיכון לזיהום נותר בין הניסוי הגדול אתגרים 20,21. למרות היתרונות הטכניים האלה של מחסניות מסננות, זה לא נעשה שימוש במחקרי עדנה של דגים עם שני חריגים 8,15. כאן אנו מספקים פרוטוקול עבור סינון של דגימות מים עם מחסנית מסנן וסחיטת עדנה ממסנן שלה מבלי לחתוך את הדיור. כמו כן אנו מספקים שתי מערכות סינון מים חלופיות תלוי כרכי מים (L ≤4 או&gt; 4 L). כדי להשוות את הביצועים של הפרוטוקול החדש שפותח ובפרוטוקול בעבר בשימוש באמצעות מסנן מזכוכית בקבוצת המחקר שלנו 12,14,22,23, אנו מבצעים עדנה metabarcoding ניתוח של מי ים מטנק אקווריום ענק (7,500 מ &#39;3 ) עם הרכב מינים, ולהראות את מספר המינים שזוהו נגזרים משני הפרוטוקולים כתוצאות נציג. h פרוטוקול זהכפי שפותחו עבור metabarcoding עדנה מן הדגים, אך גם לגבי עדנה מאורגניזמים אחרים.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Mesh panel</td>
			<td style="width:167px;">Iris Ohyama</td>
			<td style="width:119px;">MPP-3060-BE</td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Metal prong</td>
			<td style="width:167px;">Iris Ohyama</td>
			<td style="width:119px;">MR12F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stand for the mesh panel</td>
			<td style="width:167px;">No brand</td>
			<td>4184-9507</td>
			<td>available from Amazon Japan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1-L plastic bag with screw cap</td>
			<td style="width:167px;">Yanagi</td>
			<td style="width:119px;">DP16-TN1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Male luer-lock connector</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">11620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">10-mL pipette tip</td>
			<td style="width:167px;">Eppendorf</td>
			<td style="width:119px;">0030 000.765</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">10-L book bottle with valve</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-2169-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sterivex-HV filter</td>
			<td style="width:167px;">Millipore</td>
			<td style="width:119px;">SVHVL10RC</td>
			<td>denoted as &#34;filter cartridge&#34; throughout the ms and used in the protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Male luer fitting</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7379-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Female luer fitting</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">5-1043-14&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Inlet luer cap</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">VRMP6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Outlet luer cap</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">VRFP6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">High vacuum tubing</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">6-590-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Vacuum connector</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">6-663-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone stopper</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7650-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Manifold</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">2-258-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Aspirator-GAS-1</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7483-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DNeasy Blood &#38; Tissue Kit (250)</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">69506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">PowerWater Sterivex DNA Isolation Kit</td>
			<td style="width:167px;">MO BIO</td>
			<td style="width:119px;">14600-50-NF</td>
			<td>denoted as &#34;optional kit&#34; in the ms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tabletop Centrifuge</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">Model 4000</td>
			<td>Maximum speed 6,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fixed-angle rotor</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">AT-508C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adaptor for a 15 mL conical tube</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">055-1280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">RNAlater Stabilization Solution</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;"></td>
			<td style="width:167px;">Parafilm</td>
			<td style="width:119px;">PM992</td>
			<td>denoted as &#34;self-sealing film&#34;</td>
		</tr>
	</tbody>
</table>

use of a filter cartridge for filtration of water samples and extraction of environmental dna

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

מבחינה טכנית, קשה לבודד ולכמת עדנה דגים רק מן עדנה בתפזורת חילוץ, משום פריימרים MiFish coamplify באזור היעד מכמה חוליות שאינם דגים, כגון עופות ויונקים, עם מוצרי ה- PCR של באותו גודל (ca. 170 נ&quot;ב 12). במקום לכימות דגי עדנה, אנו מבצעים MiFish metabarcoding ניתוח של ...

Watch this Scientific Journal Video about Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA at JoVE.com

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

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

שימוש במחסנית סינון עבור הסינון של מי דוגמאות הפקת DNA הסביבה

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

The overall goal of this protocol is to filter water samples with a filter cartridge and extract environmental DNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms. This method can help answer key questions in the ecological field, such as biodiversity monitoring and community ecology. The main advantage of this technique is that the filter cartridges can accommodate larger water volumes and the filtration and DNA extraction can be performed in a sterile manner.To begin this procedure, attach high vacuum tubing to the input connector of an aspirator pump. Attach the other end of the tubing to a manifold. Be sure that the three red T-valves at the manifold are in the off position.Next, put on a clean set of gloves. Insert a female luer fitting at the top end of the vacuum rubber tubing. Then, insert a vacuum connector at the bottom end.Carefully attach the female luer fitting to an outlet port of the filter cartridge. Then, attach the vacuum connector to a silicone stopper. Pour one liter of sampled water into the prepared plastic bag.Enclose the screw cap with the male luer-lock connector. Then, carefully connect an inlet port of the assembled filter cartridge with the male luer-lock connector. Ensure that all connections are secure.Then, hang the plastic bag from two prongs on the mesh panel. Insert a silicone stopper into the inlet port of the manifold. Then, turn on the aspirator.Open the red T-valves for filtration. Run the manifold until the filter cartridge is dry. Then, close the red T-valves.Repeat this entire process to filter the desired amount of water. Once all of the sampled water has been filtered, carefully remove the filter cartridge from the plastic bag and vacuum container. Cap both ends of the cartridge with the appropriate luer caps.Then, using a solvent proof pen, label the cartridge and inlet luer cap. Store at 20 degrees Celsius until ready to perform DNA extraction. For samples larger than four liters, prepare a ten liter book bottle as detailed in the text protocol.Add the water sample to be filtered. Then, tightly insert a ten milliliter pipette tip into the valve of the book bottle. Tightly insert the inlet port of the filter cartridge into the pipette tip.Then, insert a silicone stopper into the inlet port of the manifold. Turn on the aspirator. Then, open the red T-valves for filtration.Run the manifold until the filter cartridge is dry. After the filtration is complete, close the red T-valves. Carefully remove the cartridge from the plastic bag and vacuum connector.Then cap both ends of the cartridge with the appropriate luer caps. Use a solvent proof pen to label the cartridge and the inlet luer cap. Store at 20 degrees Celsius until ready to perform DNA extraction.Preheat an incubator to 56 degrees Celsius. Then, cut the hinged cap from a 2.0 milliliter tube and discard the cap. Remove the inlet luer cap from the filter cartridge.Then, insert the inlet port into the 2.0 milliliter tube. Using a self-sealing film, tightly seal the connection between the cartridge and the collection tube. Next, insert the combined unit into a centrifuge adapter for a 15 milliliter conical tube.Centrifuge the cartridge for one minute at 5000 times g to remove the remaining liquid in the filter. Once centrifugation is complete, remove the collection tube from the filter cartridge and discard the tube. Then, recap the inlet port.Next, mix 20 microliters of protein A-Case solution, 220 microliters of phosphate-buffered saline, and 200 microliters of buffer AL.Remove the inlet cap and add the mixture into the cartridge using a pipette, inserting the pipette completely into the inlet port. Recap the inlet port. Then, place the filter cartridge on a rotary shaker.Place the rotary shaker in the preheated incubator, and set the shaker to 20 rpm for 20 minutes. During the incubation, label a new 2.0 milliliter tube with a solvent proof pen. Then, place it into a 50 milliliter conical tube.After the incubation is complete, remove the inlet cap. Then, insert the inlet port into the new 2.0 milliliter tube. Cap the conical tube with a screw cap.Centrifuge for one minute at 5000 times g to collect the extracted DNA. Then, remove the cartridge in the 2.0 milliliter tube using sterilized forceps. Cap the tube and purify the extracted DNA as outlined in the text protocol.In this procedure, four different volumes of sea water are filtered on the same manifold using two unique filters, and eDNA is subsequently extracted. The filter cartridge detected, on average, 50%more species than the glass fiber filters. Once mastered, this technique can be done in two hours if it is performed properly.After watching this video, you should have a good understanding of how to filter water samples with a filter cartridge, and how to extract eDNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms.

Research

JoVE Journal

Environment

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

EPA שיטת המדידה של 1615. Enterovirus ומופע norovirus במים על ידי תרבות וRT-qPCR. I. איסוף דגימות וירוסים

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

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

גישת Bioreporter תא כל להערכת תחבורה וזמינות ביולוגית של מזהמים אורגניים במערכות לא רוויים מים

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

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

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

שימוש בChironomidae (כפולי כנף) גלמי Exuviae כמהיר Bioassessment פרוטוקול לגופי מים צפים-שטח

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA שיטת המדידה של 1615. Enterovirus ומופע norovirus במים על ידי תרבות וRT-qPCR. חלק שלישי. איתור וירוסים על ידי RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

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

בחירת גודל האוטומטי ג&#39;ל כדי לשפר את האיכות של ספריות רצף הדור הבא הוכן מדגימות סביבתיות מים

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

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

שיטה פשוטה למיצוי שלב המוצק האוטומטי של דגימות מים לניתוח החיסוני של מזהמים קטנים

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

Air-sampled Filter Analysis for Endotoxins and DNA Content

Air-שנדגמו ניתוח סינון עבור אנדוטוקסינים ותוכן DNA

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

חפירה באדמה Lysimeter עבור ההידרולוגי מצמידים, גיאוכימיות, וחקירות מיקרוביולוגיות

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

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

ליפיד הפקת וניתוח שיטה לאפיון קרקע חיידקים בניסויים עם דוגמאות רבות

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

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

הפקת חומרי הדברה אורגנוכלורינים מפלסטיק וגלגול סוג פלסטיק

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