この作品は、全米科学財団環境ゲノミク​​スプログラム（グラント番号EF - 0723707）により賄われていた。

1。微生物細胞の抽出： <ol><li>徹底的に混合することにより滅菌粉砕乳棒でバイオマットをホモジナイズする。場所は約全30ワーリングブレンダーの滅菌​​容器に均質化したマットの材料のg（湿重量）、1MのNaCl（または使用中のサンプルに特定の濃度）の約100mLを追加し、1分間、中程度の速度で三回をブレンド1分間-20℃の冷凍庫で断続的に冷却。 250mLの遠心ボトルにスラリーを移し、1 M NaClで、残りの空のボリュームを埋める。オートクレーブのDI水にNaCl溶液を準備し、それを殺菌するフィルタ。 </li><li>さらに30分間室温でボルテックス（渦魔神R 2）を用いて（150 rpm）し振とうすることにより、マトリックスからの微生物細胞を取り除く。 </li><li> 4℃で15分間のための低速で遠心（500分の1グラム）ゆっくりと堆積物への最小限の障害でフラスコに上清を移す。 </li><li>ペレット堆積物を使用して、ステップ1.3の末尾に、各堆積物ペレットに新鮮なNaClの添加でブレンドし、手順1.2と1.3はさらに4回繰り返します。各NaClの抽出からの上清を新しいフラスコに転送されます。 </li><li> 4℃で15分間高速（25,000 × g）で200mLのアリコートに5セルの抽出と遠心の上清を組み合わせる℃に上清を廃棄し、2％ヘキサメタリン酸ナトリウム10mlに各細胞ペレットを再懸濁し、ペレットを組み合わせ、2％ヘキサメタリン酸ナトリウムを加えて200mlにボリュームを構成しています。 30分間室温（150 rpm）で振とうして細胞を洗浄。このステップの最後には、次のステップに進むために、細胞の唯一のチューブが必要です。 </li><li> 4℃で15分間のための高速で遠心分離（25,000 × g）でTE（50mMのEDTA）200mLに上清を、再懸細胞ペレットを捨て、そして室温で10分間（150回転）振とうして細胞を洗浄。 </li><li> 4℃で15分間、高速（25,000 × g）で遠心℃で、15 mLのTE（10mMのEDTA）に再懸濁し、上清を、破棄、および-80℃ストア200μL分注し° CまでのDNA抽出のために必要。 </li></ol> 2。 DNAの抽出と精製： <ol><li> 200μLの微生物細胞のアリコートに200μLの5 M NaCl、200μL10％SDS、および100μL14.3 Mβ-メルカプトエタノールを追加。反転4-6回で静かに混和。 </li><li> 5分間65℃の水浴で融解することによって続いて2分間液体窒素に浸して凍結融解の3ラウンドに従う混合物。 10分に解凍最後の拡張。 </li><li> 200μLの5 M酢酸カリウム（pH5.5）に、10分間氷上に場所を追加します。 4℃で10分間、10,000 xgで遠心℃、ワイドボアピペットチップを用いて上清を新しいチューブに移す。 1時間のためにRNaseを37℃でインキュベート℃の3μLを加える。 </li><li>等量のクロロホルムを追加し、反転が簡単に振る、そして室温で10分間、15,000 xgで遠心。 </li><li>慎重にワイドボアピペットチップを使用して、新しいチューブに上清の最上層を移す。上部と下部層の間にある白い界面層をピペッティングは避けてください。クロロホルム抽出の手順を繰り返します。 </li><li> DNAを沈殿に30分間氷上で上清とインキュベートするとチルドの等量が（-20℃）、イソプロパノール追加。 </li><li> 4℃で10分間ペレットDNAにCの最大速度（20800 × g）で遠心。 </li><li>上清を破棄し、チルド1mLでDNAペレットを洗浄は、70％エタノール（-20℃時）。 </li><li>で10分間4℃で最大スピード（20800 × g）で遠心機は、70％エタノールで洗浄し、10分間空気乾燥DNAペレットを繰り返し、上清を捨てる。 70％エタノールで洗浄を繰り返します。 </li><li> 25μLのTE（10mMのEDTA）に再懸濁し、DNA、65℃暖かい℃で5分間は、複数のチューブが使用されている場合は、1つのチューブに結合し、そしてTEで500μLにボリュームをメイク。 </li><li>追加500μLの氷冷（または4℃）20％ポリエチレングリコール（PEG）（1.2 M NaClに調製）。氷上で10分間インキュベート反転、、、4℃で10分間ペレットDNAにCの最大速度で遠心分離により穏やかに混合する。取り外して廃棄上清を、10分間空気乾燥、冷却した70％エタノール1mLでペレットDNAを洗浄、30〜50μlのTEまたは分子等級水に再懸濁します。 65温暖化によるDNAの再懸濁を促進〜80時5分とストアのDNAのためのC℃に</li></ol> 3。 DNAの純度、濃度とサイズの決定： <ol><li> 280分の260と光度計1000分光光度またはその他の適切な計測機器を使用して230分の260の比率を測定することにより、DNAの品質を決定します。 </li><li>メーカーの指示に従ってクワント- ITのdsDNAのアッセイキットを用いてDNA濃度を決定する。 </li><li>パルスフィールドゲル電気泳動を用いたDNAのサイズを決定します。 0.5 × TBEで1％アガロースゲルを準備し、以下のCHEF MapperのXAシステム上でパラメータ使用、0.35 sの最初のスイッチ時間、7.67秒の最後のスイッチ時間、および120℃の時6.0 V / cmのグラデーションの角度を含め線形R係数をリアンプ。 30分間1 × SYBRグリーンIを使用してゲルを染色する。ゲル上700-1000 ngのDNAの間にロードします。図を参照してください。完全なプロトコルの圧縮された形式3。 </li></ol>代表的な結果： 細胞の抽出： シーケンシャル微生物の細胞抽出物は、（上清）抽出物の濁度は、抽出数が増えるにつれて減少することが示されている。これは、連続したセルの抽出後の細胞数の減少を示唆している。ここで注意すべき重要な追加の各セルの抽出工程は、汚染物質の導入のための機会を提供するので、細胞の抽出数は、最終細胞ペレットは、全体的な微生物群集の代表となるように最小化されるべきであるということです。汚染の他の情報源は、適切な実験室無菌技術を採用することによって制御されていました。例えば、ソリューションは、オートクレーブ純水で調製し、滅菌フィルター。容器は、アルコールで滅菌オートクレーブ、紫外線や紫外線架橋剤で処理した。 DNAの濃度と品質決定： プロトコルは、マットの1.6の280分の260比を持つ試料、および0.7の230分の260の比（表1）のグラムあたりHMW DNAの約2μgの（35〜50キロバイト）（図4）を得た。 230分の260の比率が低いように見えるが、そのような16S rRNA遺伝子のPCR -増幅のような下流分子ベースのアプリケーションの阻害は別の研究7で観察されなかった。 EPSと塩;塩性マットからそのDNAコンタミネーションの2つの主な情報源を持っていることに注意することが重要です。それは、これらの汚染物質の痕跡がダウンストリームのアプリケーションに与える影響を低減するために多大な努力にもかかわらず、230分の260の比率に影響を与えることができることが可能となる。 DNAサイズの決定： 図3に示すように、パルスフィールドゲル電気泳動は、DNAの塗抹標本で、その結果、断続的な方向のスイッチと脈動​​電流の流れを採用しています。我々のプロトコルは、約35〜50キロバイトのHMW DNAが得られた。いくつかのDNAサイズが30 KBより小さいかもしれないが、それは、フォスミドクローン作成は、約40 kbのDNAを挿入し、より大きなDNA断片は、無傷の生合成経路へのより大きなアクセスを提供する必要があることを考えると35キロバイト上記の塗抹標本の一部を持つことが重要です。我々の研究では、35キロバイト上記のDNAスミアは大インサートベクトルのクローニングと他の分子のアプリケーション用に切り出し、精製した。 <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="図1"/> エリューセラ、バハマにある図1。塩性の微生物マットのサンプリングサイト（ビッグ池）。 <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="図2"/> 図2本研究で使用した塩性微生物マットの断面。微生物マットはエリューセラ、バハマにある塩性池から入手した。 <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="図3"/> 図3。微生物細胞の抽出、細胞溶解、抽出、およびメタゲノムDNAの精製に関連する手順の模式図は。 <a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">拡大画像を表示するにはここをクリックしてください。</a> <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="図4"/> 図4パルスフィールドゲル電気泳動を用いて抽出したDNAの分子量特性評価。レーンMは、HMWマーカーであり、レーン1と2は、上記のプロトコルを使用してエリューセラ塩性のマットから抽出したメタゲノムDNAの複製です。 <table border="1"><tbody><tr><td> 抽出方法 </td><td></td><td> 濃度ng / gの </td><td> 280分の260 </td><td> 230分の260 </td></tr><tr><td> PEG </td><td>担当者1 </td><td> 1806.1 </td><td> 1.64 </td><td> 0.76 </td></tr><tr><td></td><td>担当者2 </td><td> 2010.8 </td><td> 1.61 </td><td> 0.75 </td></tr></tbody></table> 表1。微生物塩性マットから抽出したDNAの濃度と品質の測定。

<ol>
	<li>Decho, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms+in+intertidal+systems:+an+overview.">Microbial biofilms in intertidal systems: an overview.</a> Cont. Shelf Res. 20, 1257-1273 (2000).</li><li>Dupraz, C., Visscher, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+lithification+in+marine+stromatolites+and+hypersaline+mats.">Microbial lithification in marine stromatolites and hypersaline mats.</a> Trends Microbiol. 13, 429-438 (2005).</li><li>Steffan, R. J., Goksoyr, J., Boj, A. K., Atlas, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+from+soils+and+sediments.">Recovery of DNA from soils and sediments.</a> Appl. Environ. Microbiol. 54, 2908-2915 (1988).</li><li>Lee, Y. K., Kim, H. W., Liu, C. L., Lee, H. K. <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+method+for+DNA+extraction+from+marine+bacteria+that+produce+extracellular+materials.">A simple method for DNA extraction from marine bacteria that produce extracellular materials.</a> J. Microbiol. Methods. 52, 245-250 (2003).</li><li>Roose-Amsaleg, C. L., Garnier-Sillam, E., Harry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+Purification+of+Microbial+DNA+from+Soil+and+Sediment+Samples.">Extraction and Purification of Microbial DNA from Soil and Sediment Samples.</a> Appl. Soil Ecol. 18, 47-60 (2001).</li><li>de Lipthay, J. R., Enzinger, C., Johnsen, K., Aamand, J., S&#248;rensen, 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=Impact+of+DNA+extraction+method+on+bacterial+community+composition+measured+by+denaturing+gradient+gel+electrophoresis.">Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis.</a> Soil Biol. Biochem. 36, 1607-1614 (2004).</li><li>Bey, B. S., Fichot, E. B., Dayama, G., Decho, A. W., Norman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+High+Molecular+Weight+DNA+from+Microbial+Mats.">Extraction of High Molecular Weight DNA from Microbial Mats.</a> Biotechniques. 49, 631-640 (2010).</li><li>Kakirde, S. K., Parsley, L. C., Liles, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+does+matter:+Application-driven+approaches+for+soil+metagenomics.">Size does matter: Application-driven approaches for soil metagenomics.</a> Soil Biology &#38; Biochemistry. 42, 1911-1923 (2010).</li><li>Rodon, M. R., August, P. R., Bettermann, A. D., Brady, S. F., Grossman, T. H., Liles, M. R., Loiacono, K. A., Lynch, B. A., MacNeil, I. A., Minor, C., Tiong, C. L., Gilman, M., Osburne, M. S., Clardy, J., Handelsman, J., Goodman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+the+Soil+Metagenomic:+A+Strategy+for+Accessing+the+Genetic+and+Functional+Diversity+of+Uncultured+Microorganisms.">Cloning the Soil Metagenomic: A Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms.</a> Appl. Environ. Microbiol. 66, 2541-2547 (2000).</li><li>Beja, O., Aravind, L., Koonin, E. V., Suzuki, M. T., Hadd, A., Nguyen, L. P., Jovanovich, S. B., Gates, C. M., Feldman, R. A., Spudich, J. L., Spudich, E. N., 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=Bacterial+Rhodopsin:+Evidence+for+a+New+Type+of+Phototrophy+in+the+Sea.">Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea.</a> Science. 289, 1902-1906 (2000).</li></ol>

利害の衝突は宣言されません。

複雑かつ高度に多様な微生物マット試料からの全細胞の除去が実用的ではないことを考えると、主な関心事は、抽出された細胞は、全体的な微生物マットのコミュニティを表す方法もあります。以前の研究で、微生物の16S rRNA遺伝子のPCR - DGGE解析では、このプロトコルで使用する5つの細胞の除去の手順は、全体的な微生物マットのコミュニティ7の代表的な細胞を抽出することを示し...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td> β-メルカプトエタノール</td><td>シグマアルドリッチ</td><td> M3148 </td><td></td></tr><tr><td>ポリエチレングリコール8000 </td><td>プロメガ</td><td> V3011 </td><td> 1.2 M NaClを約20％ </td></tr><tr><td>カリウム酢酸</td><td>フィッシャーサイエンティフィック</td><td></td><td>フィッシャーサイエンティフィック</td></tr><tr><td>クワント- IT dsDNAをアッセイキット</td><td>インビトロジェン</td><td> Q33130 </td><td></td></tr><tr><td> RNaseの</td><td> EPICENTRE </td><td> MRNA092 </td><td></td></tr><tr><td>塩化ナトリウム</td><td> BDHケミカルズ</td><td> BDH8014 </td><td>適切なコンク。 </td></tr><tr><td>硫酸ドデシルナトリウム</td><td>フィッシャーサイエンティフィック</td><td> 03-500-509 </td><td>水の10％ </td></tr><tr><td>ヘキサメタリン酸ソーダ</td><td> EMDケミカルズ</td><td> SX0583 - 3 </td><td>水の2％ </td></tr><tr><td> TBE </td><td>フィッシャーサイエンティフィック</td><td> BP1333 - 1 </td><td></td></tr><tr><td> CHEF MapperのXAシステム</td><td>バイオラッドラボラトリーズ</td><td> 170-3670 </td><td></td></tr><tr><td>光度計1000分光</td><td>サーモサイエンティフィック</td><td> ND - 1000 </td><td></td></tr><tr><td>ボルテックス</td><td>サイエンティフィックインダストリーズ株式会社</td><td></td><td></td></tr><tr><td>紫外線架橋剤</td><td> UVP </td><td></td><td></td></tr><tr><td>ワーリングブレンダー</td><td>ワーリング実験室</td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

メタゲノムデータの成功したと正確な分析と解釈は、質の高い、高分子量（HMW）のコミュニティDNAの効率的な抽出に依存しています。しかし、環境マットのサンプルは、しばしば高品質の、HMWのDNAの大規模な濃度を得るために困難をもたらす。塩性の微生物マットは高い細胞外高分子物質の量（EPS）1、抽出されたDNAの下流のアプリケーションを阻害する塩が含まれています。ダイレクトと厳しい方法は、しばしば難治性の試料からのDNA抽出に使用されています。マット、接着剤マトリックス、のEPSは直接溶解中にDNA 2,3を結合するので、これらのメソッドは一般的に使用されています。厳しい抽出方法の結果として、DNAは、小さいサイズ4,5,6に分割になります。 DNAは、このように大規模挿入ベクトルのクローニングには不適切となります。これらの制限を回避するために、我々は塩性微生物マットから良い質と量のHMW DNAを抽出するために改良された方法論を報告する。我々はブレンドと分画遠心法を通じて、背景のマットマトリックスからの微生物細胞の分離を伴う間接的な方法を採用。機械的および化学的手順の組み合わせは、抽出された微生物細胞からDNAを抽出精製するために使用された。我々のプロトコルは1.6の280分の260比で、マットのサンプルのグラム当たりHMW DNA（35〜50キロバイト）の約2μgを得られます。また、16S rRNA遺伝子7の増幅は、プロトコルが汚染物質のいずれかの阻害効果を最小化または排除することができることを示唆している。我々の結果は、機能的なメタゲノム研究のためのバイオマットからHMWのDNAの抽出のための適切な方法論を提供し、DNA抽出が難しいされているから、他の環境試料にも適用可能である。

Watch this Scientific Journal Video about Extraction of High Molecular Weight DNA from Microbial Mats at JoVE.com

Extraction of High Molecular Weight DNA from Microbial Mats

我々は、塩性微生物マットから高分子量のDNAを抽出するための改良されたプロトコルを提供しています。微生物細胞は、DNAの抽出と精製の前にマットのマトリックスから分離されています。これは、濃度、品質、およびDNAの大きさを高めます。プロトコルは、他の難治性のサンプルに使用することができる。

バイオマットから高分子量DNAの抽出

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

extraction-of-high-molecular-weight-dna-from-microbial-mats

Research

JoVE Journal

Biology

19.4K ビュー.  Arnold School of Public Health, University of South Carolina. 我々は、塩性微生物マットから高分子量のDNAを抽出するための改良されたプロトコルを提供しています。微生物細胞は、DNAの抽出と精製の前にマットのマトリックスから分離されています。これは、濃度、品質、およびDNAの大きさを高めます。プロトコルは、他の難治性のサンプルに使用することができる。

ビデオ: Extraction of High Molecular Weight DNA from Microbial Mats

Biology of Microbial Communities  - Interview

微生物群集の生物学 - インタビュー

生物学

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

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

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

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

0.22μmのSterivexフィルタおよび塩化セシウム密度勾配遠心分離からのDNA抽出

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

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

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

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

土壌と堆積物から高分子量のゲノムDNAの抽出

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

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

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

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

遺伝学的およびエピジェネティクス解析のためのパラフィン包埋材料からのDNA抽出

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

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.

家禽生産のサンプルからの細菌群集の定性的および定量的評価のためのハイブリッドDNA抽出法

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

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

ムチンアガロースゲル電気泳動：高分子量の糖タンパク質のためのウェスタンブロッティング


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

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及びDNA抽出の両方のためのホルマリン固定パラフィン包埋組織コアの調製

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

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

高スループットDNA抽出プレートの装填のための改変方法により、汚染の可能性を低減

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

カエノラブディティス・エレガンスゲノムDNAの小規模抽出

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...