Yenilik, British Columbia Bilgi Kalkınma Fonu ve Ulusal Bilimler ve Mühendislik Araştırma Konseyi (NSERC) Kanada Kanada Vakfı Biz, kıyı ve açık okyanus sularında düşük oksijen bölgelerde devam eden çalışmalar destek için teşekkür etmek istiyorum. JJW NSERC ve DAW burs tarafından desteklenen NSERC, Killam ve TULA vakıf Mikrobiyal Çeşitlilik ve Evrim finanse Merkezi burs tarafından desteklenmiştir. SL Mikrobiyal Çeşitlilik ve Evrim TULA vakıf tarafından finanse edilen Merkezi tarafından desteklendi.

Not: Bu protokolde kullanılan tüm reaktifler çalışma hisse senetleri bu DNA örnekleri ve laboratuar stoklarının kirlilik çapraz bulaşmayı önlemek için çok önemli küçük laboratuvar stokları aliquoted. Ayrıca, pipetleme, çapraz kontaminasyonu engellemek için her türlü ek için pipet uçları değiştirmek için emin olun. Part One: Hücre Lizis ve Sindirim <ol><li> Bu protokol, buz üzerinde önceden konsantre planktonik biyokütle içeren Sterivex filtreler çözülme başlayın. Basitlik için, burada ayrı bir filtre işleme prosedürü tarif. Uygulamada, bir defada işlem 16 veya daha az filtreleri tavsiye ederiz. </li><li> Filtre çözülmüş sonra, ilk iki inkubasyon başlar: biri olmuş akyuvarlar ve proteinleri yıkmak için, RNA, ve bir kaldırmak için. Ilk kuluçka dönemi için 100 ul lizozim (1000 ul TE 125 mg) ve 20 ml RNaz A (10 mg / ml), filtre ekleyin. Parafilm ile filtre Reseal ve hibridizasyon fırında ° C &#39;de 1 saat süreyle 37 ° döndürmek için bırakın. </li><li> Sonraki kuluçka için 100 ul proteinaz K ve 100 ul% 20 SDS, filtre ekleyin. 55 Reseal hibridizasyon fırında 1-2 saat daha fazla döndürmek için Parafilm kullanarak ve bırakın bu kez ° C </li><li> 5cc bir şırınga kullanılarak, 15 ml Falcon tüp içine Sterivex filtre lizat aktarın. 1 ml lizis tamponu ile filtre durulayın ve Falcon tüp içinde lizat 5cc şırınga ile sıvı durulama ekleyin. Şimdi size parçalanmış ve hücreleri sindirilir olduğunu, onların DNA ayıklamak için hazır. </li></ol> Bölüm İki: DNA Ekstraksiyon <ol><li> Bir sonraki adım sizin lizat DNA ayıklamak için fenol-kloroform yöntemi kullanmaktır. Çapraz kontaminasyonu engellemek için her ek için pipet uçları değiştirmek için emin olun, lizat tüp izoamil Alkol (IAA): Kloroform: Fenol eşit hacmi (3ml hakkında) ekleyin. 10 saniye iyice karıştırın vorteksleyin. </li><li> 5 dakika 2500 g santrifüj dengeli olduğundan emin Spin tüp. Alt örnekten fenol, kloroform ve protein içeren bir organik katmanı, DNA, su, sulu bir tabaka, ve diğer: santrifüj sırasında, fenol-kloroform-lizat karışımı iki kat ayırmalıdır üstüne daha hidrofilik molekülleri. </li><li> Sulu tabaka (DNA içeren) yeni bir 15 ml Falcon tüp içine aktarın. Pipet ucu (her zaman arkasında sulu tabakanın küçük bir miktar terk) ile arayüz dokunmamaya dikkatli olun. </li><li> Çapraz kontaminasyonu engellemek için her ek için pipet uçları değiştirmek için emin olun, IAA: Bu yeni tüp, Kloroform eşit hacmi (yaklaşık 3 ml) ekleyin. 10 saniye karıştırın. Bu adım, DNA örneği kalan herhangi bir fenol giderilmesine yardımcı olur. </li><li> Sulu açıktır kadar 5 dakika veya 2500 g dönerler. Yeni, etiketli Falcon tüp içine aktarın ve sulu tabaka, pH 8.0 TE 1 ml pipet ucu (her zaman arkasında sulu tabakanın küçük bir miktar terk) ile arayüz dokunmamaya dikkat ederek ekleyin. Bu sulu DNA özü içerir. </li></ol> Bölüm Üç: DNA Konsantrasyon ve Yıkama <ol><li> Bir sonraki adım, 15 ml Amicon Ultra santrifüj tüpüne DNA örneği yıkayın ve konsantre. Amicon tüpleri (süzüntü), akışı yakalamak için belirli bir molekül ağırlığına (retentate) ve bir tüp ya da yukarıda molekülleri tutar bir filtre oluşur. Bu durumda, filtre DNA (retentate) korur. Adım 2.5 DNA özü bir Amicon Ultra tüp filtre bölmesine aktarın. </li><li> 3500 g de 10 dakika boyunca dönerler. Amicon filtre muhafaza sıvı az 1 ml olduğundan emin olmak için kontrol edin. Daha retentate kalır, TE filtre dolum ve tekrar döndürebilirsiniz. Çapraz kontaminasyonu engellemek için her TE ek için yeni bir pipet kullandığınızdan emin olun. </li><li> Başka bir Falcon tüp akış yoluyla veya süzüntü çıkarın ve bir jel üzerinde son DNA ürün doğruladı kadar (Dördüncü Bölüm) buzdolabında saklayın. </li><li> 2 ml TE tampon Amicon filtre ekleyin ve çapraz kontaminasyonu engellemek için her TE ek için yeni bir pipet kullanmak için emin olun, 6 dakika için 3500 g döndürün. Havuz adım 3.3 ile süzüntü ve buzdolabında saklayın. </li><li> Üç yıkar (her zaman taze bir pipet kullanmak için emin olun) toplam TE DNA iki kez daha yıkayın ve (3.3 ve 3.4 gibi adım başı) süzüntü her zaman tasarrufu. Son yıkama, sıkma Amicon filtre retentate kalıntıların 200 ila 500 ml kadar. Son ses kaydetme ve etiketli 1.5 ml Eppendorf tüp (DNA içeren) retentate aktarmak. </li></ol> Dördüncü Bölüm: DNA Kalitesinin Belirlenmesi <ol><li> DNA kalitesini kontrol edin ve bir jel% 0.8 agaroz jel sta örnekleri tükeniyor DNA konsantrasyonu tahmingecede 1ml ethidium bromür (EtBr) ile incelendiği. (Kullanırken son derece dikkatli olun EtBr bilinen bir mutajen ve şüpheli kanserojen eldivenler sık ​​sık değiştirmek ve laboratuar geri kalanına ve cildinize EtBr transferini önlemek için emin olun.) Yük görevi, DNA merdivenler yanında örnekleri, büyüklüğü ve yoğunluğu standartları (sonraki adıma bakın). </li><li> İlk birkaç şeritli, çeşitli molekül ağırlıkları ve konsantrasyon bantları ile merdivenler yük. Ilk ve son şerit (dıştaki şerit), 10 ul 1KB 50ng/μl, yük + veya 2log merdiven: Biz aşağıdaki yükleme deseni öneririz. 2,3 ve 4 şeritli yükü 2 ul, 5, ul ve 50ng/μl λHindIII merdivenin sırasıyla 10 ul,. Son olarak, her bir örnek için boya ile DNA özü her kulvar için 5 ul yükleyin. </li><li> Jel, yaklaşık 16 saat için 15 volt (gece çalıştırmanızı öneririz) çalıştırın. </li><li> Ertesi gün, bir UV jel dokümantasyon sistemi kullanılarak jel fotoğrafı. DNA ve konsantrasyonu moleküler ağırlığı aralığı belirlemek için örnek bantları, merdiven bantları karşılaştırın. İyi kalitede DNA yüksek molekül ağırlıklı (&gt; 36KB) var ve çok az kanıt kesme / bozulma gösterecektir. Bkz: Şekil 1. </li></ol> Beşinci Bölüm: Sezyum Klorür Gradient Santrifüj <ol><li> DNA kalitesi iyi ve miktarı yeterli değilse, bir sezyum klorür (CSCL) gradient santrifüj yaparak DNA arındırmak için devam edebilirsiniz. Bu arıtma adım isteğe bağlıdır ve downstream uygulamalar (örneğin, sadece PCR reaksiyonları gerçekleştirmek istiyorsanız ise, arıtma genellikle gerekli değildir, arındırmak GEREKEN fosmid kütüphaneleri oluşturmak istiyorsanız) için gerekli olduğunu unutmayın. Öncelikle, her bir örnek için bir santrifüj tüpüne etiket. </li><li> Her bir santrifüj tüpüne, 160 CSCL mg, 178 ul genomik DNA ekleyin. Tüp CSCL ve DNA ekledikten sonra, parafilm tüpün üstüne küçük bir parça koyun ve bileşenleri karıştırmak için hafifçe tüp on ila yirmi kez ters çevirin. Pipetleme ile karıştırmayın, DNA kesme neden olabilir. Sonra bu tüp, 10 mikrogram / ul EtBr 10 ul aynı şekilde ekleyin ve karıştırın. (Kullanırken son derece dikkatli olun EtBr bilinen bir mutajen ve kanserojen şüpheli. Eldivenler sık ​​sık değiştirmek ve laboratuar geri kalanına ve cildinize EtBr transferini önlemek için emin olun.) </li><li> Tüpler arasındaki ağırlık farkı 1 mg daha az olduğu gibi santrifüj tüpleri ağırlığı önce dengeli olduğundan emin olun. </li><li> Rotor kullanarak hemostat tüplerin yerleştirin, kapağını kapatın ve ultrasantrifüjdeki içindeki rotor. </li><li> Ultrasantrifüjdeki kapağını kapatın. Vakum kapıya yakın olarak en kısa sürede yapılacaktır. 18 saat boyunca 100.000 rpm ve 20 ° C gece çalıştırın </li><li> Santrifüj tamamlandığında, bir tüp raf hemostat ve yer kullanarak rotor tüpleri almak. </li><li> Mavi ışık transilluminator&#39;de ile DNA görselleştirin veya kullanılamaz, karanlık bir odada, uzun dalga boylu UV ışığı kullanın. DNA bant görmek için kehribar filtre gözlük bir çift giyin. Bakınız Şekil 2. </li><li> 1 cc steril şırınga ve iğne (26g 5 / 8) kullanarak, DNA bandı çıkarın ve bir 1.5ml Eppendorf tüp içine koyun. </li><li> Şırınga içinde ölü bir alan içinde sıkışıp DNA en kurtarmak yardımcı olmak için, 100 ul TE şırınga ile yıkayın ve tüp durulanır çözüm ekleyin. Çapraz kontaminasyonu önlemek için bir tüp numune başına 100 ul TE tampon ile hazırlayın. </li><li> DNA EtBr kaldırmak için, yavaşça borular on ila yirmi kez, 1 dakika 10.000 rpm&#39;de santrifüj invert tüpüne eşit hacimde su doymuş bütanol eklemek ve üst tabaka atın. </li><li> - 4 kez daha bütanol renk, 3 şeffaf kadar yıkama işlemi tekrarlayın. </li><li> Place 4 ml bir Amicon Ultra santrifüj tüpüne TE ve yukarıda DNA çözüm ekleyin. </li><li> DNA hacmi yaklaşık 100-500 ul (yaklaşık 6-7 dakika) azalır kadar, 3500 g oda sıcaklığında santrifüj ve flow-through atın. </li><li> 2ml TE Amicon filtre ekleyin ve çapraz kontaminasyonu engellemek için her ek için yeni bir pipet kullanmak için emin olun, 6 dakika için 3500 g santrifüj. Iki kez tekrarlayın. </li><li> Konsantre gerekli gibi ek santrifüj 50-100 ul son bir hacmi ve yeni bir 1,5 ml Eppendorf tüp filtre DNA çözümü transfer. </li><li> 40 ul TE Amicon filtre ekleyin ve kalan herhangi bir DNA yıkamak için her iki filtre membranlar boyunca yukarı ve aşağı pipetle. Bu çözüm, 5.15 tüp ekleyin. </li><li> 200 ul otoklava su ekleyerek ve 7 dakika 10.000 rpm&#39;de santrifüj Microcon tüp ve ön yıkama Microcon Microcon YM-30 filtre ünitesi yerleştirin. </li><li> DNA daha fazla konsantre 5.16 den DNA çözümü amacıyla prewashed Microcon ekleyin. 1 - 3 dakika 5000-10000 g santrifüjleyin. Sıvı miktarını kontrol edin, filtre ve tekrar santrifüj filtre üzerinde sıvı miktarı yaklaşık 50 ul azalır kadar. </li><li> , Yeni bir Microcon tüp içinde baş aşağı filtre ünitesi koyun ve 3 dakika 1000 g santrifüj. Konsantre çözüm ideal miktar 50-60 ul. </li><li> Ve bir Nanodrop DNA konsantrasyonu ölçün, kayıt ve zirve kalite kontrol (260nm olmalıdır). </li><li> DNA küçük bir kısım eksi 20 hisse senedi ve dondurma çalışmak için temiz bir Eppendorf tüp aktarın. DNA geri kalanı, eksi 80, ikinci bir temiz Eppendorf ve donma aktarın. </li></ol> Temsilcisi Sonuçlar: Bu protokol doğru yapıldığında, Şekil 1 &#39;e benzer DNA kalitesinin belirlenmesi 4.4 adım sonra bir jel görüntü göreceksiniz. Gerçek DNA konsantrasyonu özler örnek kaynağına bağlı olarak değişir. CSCL degrade santrifüj 5.7 adım sonra, mavi ışık ile aydınlatılan genomik DNA Şekil 2 &#39;e benzer bakmak gerekir. <img src="/files/ftp_upload/1352/1352Fig1newer.png" alt="Şekil 1" /> Şekil 1 intercalating ajan etidyum bromür (10 mg / ml) ile boyandı güney kutup dairesine Pasifik Okyanusu (iki nüsha), dört derinliklerinde toplanan yüksek molekül ağırlıklı DNA% 0.8 agaroz jel elektroforezi görüntü. Jel 1X TBE jel çalışan tampon için 15V ~ 16 saat çalıştırıldı. Örnek bantları mekanik kesme (birden fazla bantları karşı tek bantlar veya smear gösterir) çok az kanıt gösteren kaliteli 10m özleri korumak rağmen bazı RNA (0,5 ila 2,0 Kb aralığında smear) üzerinde taşır. <img src="/files/ftp_upload/1352/1352Fig2.jpg" alt="Şekil 2" /> Şekil 2 CSCL degrade santrifüj sonra mavi ışık ile aydınlatılan Genomik DNA bant.

<ol>
	<li>Beja, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+BAC+or+not+to+BAC:+marine+ecogenomics.">To BAC or not to BAC: marine ecogenomics.</a> Curr Opin Biotechnol. 15 (3), 187-190 (2004).</li><li>Beja, O., Suzuki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+analysis+of+bacterial+artificial+chromosome+libraries+from+a+marine+microbial+assemblage.">Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage.</a> Environ Microbiol. 2 (5), 516-529 (2000).</li><li>DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community+genomics+among+stratified+microbial+assemblages+in+the+oceans+interior.">Community genomics among stratified microbial assemblages in the oceans interior.</a> Science. 311, 496-503 (2006).</li><li>Stein, J. L., Marsh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+uncultivated+prokaryotes:+isolation+and+analysis+of+a+40-kilobase-pair+genome+fragment+from+a+planktonic+marine+archaeon.">Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.</a> J Bacteriol. 178 (3), 591-599 (1996).</li><li>Somerville, C. C., Knight, I. T., Straube, W. L., Colwell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+rapid+method+for+the+direct+isolation+of+nucleic+acids+from+aquatic+environments.">A simple, rapid method for the direct isolation of nucleic acids from aquatic environments.</a> Appl. Environ. Microbiol. 55 (3), 548-554 (1989).</li></ol>

Işlenecek kaç örneklerindendir bağlı olarak, bu iki saatlik inkübasyon adımları ve tekrarlanan yıkama ve santrifüj adımları nedeniyle bir süre yoğun bir prosedür olabilir. Bol zaman terk etmek, bu işlem için iki tam gün planlamak için en iyisidir. Artık uygun hacmi kadar (Bölüm Üç tekrarlayın) ek TE yıkar ve santrüfüj yapmaya çalışın, DNA konsantrasyonu ve yıkama sırasında 200-500μl azaltmak için Amicon tüp nihai özü hacmi sorunları vardır. Ayrıca, eğer özü akut kloroform kokuyor, kokusu tüm kloroform kaldırılır emin olmak için azaltır kadar ek yıkar yapmak en iyisidir. Yine, bu protokolde kullanılan tüm reaktifler küçük çalışma içine laboratuvar stokları aliquoted hisse senetleri bu DNA örnekleri ve laboratuar stoklarının kirlilik çapraz bulaşmayı önlemek için çok önemlidir. Ayrıca, pipetleme, çapraz kontaminasyonu engellemek için her türlü ek için pipet uçları değiştirmek için emin olun.

A correction was made to "<a href="/video/1352">DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation</a>". A typo in a volume's measurement was corrected in step 1.2.
The step was corrected from:
Once the filter has thawed, begin the first of two incubations: one to lyse the cells and remove RNA, and one to break down proteins. For the first incubation, add 100 &#956;l lysozyme (125 mg in 1000 &#956;l TE) and 20 ml RNase A (10 &#956;g/ml) to the filter. Reseal the filter with Parafilm and leave it to rotate in the hybridization oven at 37&#176;C for 1 hour.
to:
Once the filter has thawed, begin the first of two incubations: one to lyse the cells and remove RNA, and one to break down proteins. For the first incubation, add 100 &#956;l lysozyme (125 mg in 1000 &#956;l TE) and 20 &#956;l RNase A (10 &#956;g/ml) to the filter. Reseal the filter with Parafilm and leave it to rotate in the hybridization oven at 37&#176;C for 1 hour.

A correction was made to <a href="/video/1352">DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation</a>. A typo in a volume's measurement was corrected in step 1.2.

Erratum: DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 cc syringe (26G5/8 needle)</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309597</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe (26G5/8 needle)</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309597</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml eppendorf tubes</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>0030 125.150</td>
<td>&nbsp;</td>
<tr>
<td>15ml tubes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>CLS430791</td>
<td>&nbsp;</td>
<tr>
<td>5cc syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309603</td>
<td>&nbsp;</td>
<tr>
<td>Amicon&reg; Ultra-4 Centrifugal Filter Devices</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC801096</td>
<td>10,000 Nominal molecular weight limit</td>
</tr>
<tr>
<td>Butanol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A383-1</td>
<td>Make it water saturated (butanol:water = 1:1)</td>
</tr>
<tr>
<td>Centrifuge rotor, JA5.3 </td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>368690</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge, Avanti&reg; J-E</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>369003</td>
<td>&nbsp;</td>
<tr>
<td>Cesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4036</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform:IAA (24:1)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>25666-500ML</td>
<td>&nbsp;</td>
<tr>
<td>Ethidium Bromide (EtBr)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E1510-10ml</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E5134</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization oven</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-247-10</td>
<td>37&deg;C & 55&deg;C</td>
</tr>
<tr>
<td>Lysis Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3)</td>
</tr>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L6876-5G</td>
<td>125 mg in 1000 &mu;l TE</td>
</tr>
<tr>
<td>Microcon&reg; Ultracel YM-30 Centrifugal Filter Unit</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>42410</td>
<td>50 bp cut off</td>
</tr>
<tr>
<td>Parafilm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-374-10</td>
<td>&nbsp;</td>
<tr>
<td>Phenol:Chloroform:IAA (25:24:1)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>77617-500ML</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>19133</td>
<td>&nbsp;</td>
<tr>
<td>RNase A</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP2539-100</td>
<td>10 &mu;g/ml</td>
</tr>
<tr>
<td>Safe Imager&trade; Blue light transilluminator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S37102</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Dodecyl Sulfate (SDS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4509</td>
<td>20% SDS</td>
</tr>
<tr>
<td>Sterivex filters</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SVG010RS</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0389</td>
<td>&nbsp;</td>
<tr>
<td>Table top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5415D</td>
<td>&nbsp;</td>
<tr>
<td>TE buffer, pH 8.0</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trizma&reg; base</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge rotor, TLA 100</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>343847</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tube (7X20mm, PC)</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>343775</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tube rack</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>348302</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge, Optima&reg; Max</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dna extraction from 0.22 &mu;m sterivex filters and cesium chloride density gradient centrifugation

Bu yöntem, depolama / lizis tamponu ile tedavi edilen ve -80 ° C&#39;de arşivlendi 0.22 mcM Sterivex filtreler üzerinde yoğunlaştı planktonik biyokütleden yüksek molekül ağırlıklı genomik DNA ayıklamak için, ve sezyum klorür yoğunluk gradiyenti kullanarak bu DNA arındırmak için kullanılır. Protokol, hücrelerin DNA kurtarmak ve RNA kaldırmak için iki kez birer saat inkübasyon adımlar ile başlar. Sonra, bir dizi fenol: kloroform ve kloroform ekstraksiyon, proteinler ve hücre zarı bileşenleri, sulu DNA özü toplama, ve özü yıkayın ve konsantre birkaç tampon değişim adımları kaldırmak için santrifüj yoluyla takip yapılır. Beşinci Bölüm, isteğe bağlı arıtma sezyum klorür yoğunluk gradiyenti ile açıklanmıştır. Bu karışıklığı önlemek ve protokol süresini azaltmak için tek seferde en az 15 örnek ile çalışmak tavsiye edilir. Bu protokol için gerekli toplam süre çıkarılan numune sayısı bağlıdır. 10-15 örnekler ve uygun santrifüj ekipmanları mevcut olduğu varsayılarak, tüm bu protokol, 3 gün sürmesi bekleniyor. Hibridizasyon fırınlar sürecin başında ısıya ayarlanmış olduğundan emin olun.

Watch this Scientific Journal Video about DNA Extraction from 0.22 ÃŽÂ¼M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation at JoVE.com

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

Biz sezyum klorür yoğunluk gradiyenti santrifüj arıtma için 0.22 mikron Sterivex filtreler, konsantre planktonik biyokütle yüksek molekül ağırlıklı genomik DNA çıkarılması için bir metodu tanımlar.

0.22 mcM Sterivex Filtreler ve Sezyum Klorür Yoğunluk Gradient Santrifüj DNA Ekstraksiyon

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

dna-extraction-from-022-m-sterivex-filters-cesium-chloride-density

Nanyang Technological University

Research

JoVE Journal

Biology

29.0K Görüntüleme.  University of British Columbia - UBC. Biz sezyum klorür yoğunluk gradiyenti santrifüj arıtma için 0.22 mikron Sterivex filtreler, konsantre planktonik biyokütle yüksek molekül ağırlıklı genomik DNA çıkarılması için bir metodu tanımlar.

Video: DNA Extraction from 0.22 μM Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Topraklar ve tortulları Yüksek Moleküler Ağırlık Genomik DNA çıkarımı

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Biyoloji

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Genetik ve epigenetik Analizleri Parafin Gömülü Malzeme DNA Ekstraksiyon

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

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Mikrobiyal Paspas Yüksek Moleküler Ağırlıklı DNA çıkarımı

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

Ekstraksiyon ve İnsan ve Maymun Saç gelen Kortizol Analizi

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

Tesisinden Akışkan apoplastik çıkarımı için infiltrasyonu-santrifüj Tekniği Kullanarak bırakır<em&gt; Phaseolus vulgaris</em&gt; Bir örnek olarak

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

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

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Kanatlı Üretim Örneklerden Bakteriyel Topluluklar Nitel ve Nicel Değerlendirme Karma DNA Ekstraksiyon Yöntemi

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

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviral Entegrasyon Siteleri Amplifikasyon, Yeni Nesil Sıralama ve Genomik DNA Haritalama

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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

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

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

Nükleik Asitlerin süzülmesi İzolasyonu: basit ve hızlı DNA izolasyonu,

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

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

RNA ve DNA Ekstraksiyon hem Formalin sabit Parafin gömülü Doku Çekirdek hazırlanması

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Sperm yoğunluğu degrade Santrifüjü kullanarak fark kalitesini topluluğu

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...