私たちの研究室での作業は、NIGMS - NIHから国立科学財団とAREA助成R15GM86839からキャリア賞MCB0551798によってサポートされています。

1。成長とUV照射。 <ol><li> 0.4％グルコース、0.2％カザミノ酸、および10μg/ mlのチミン（DGCthy媒体）と100μg/ mlのアンピシリンを補充デイビス培地1で栽培プラスミドpBR322を含有する新鮮な一晩培養液200μlのはペレットです。細胞ペレットを、アンピシリンを欠く200μlのDGCthy培地に再懸濁し、DGCthy培地20 mlに接種するために使用されます。 </li><li>培養物は、0.5のOD 600（〜5 × 10 8細胞/ ml）に37℃振盪培養器でアンピシリン選択° Cなしで栽培されています。アンピシリンのない成長は、いくつかの変異体で発生する可能性がある異常なまたは非生産的な複製の中間体に対する選択を回避することができます。損傷を誘導するためにUV光を使用している場合は、UVの効果的な投与量を減らすこと、これらの波長とシールド細胞で強く吸収するので、さらに、、メディアからのアンピシリンの除去が必要です。 </li><li>黄色のライトの下で作業し、培養は、攪拌用の回転台に15センチメートル直径のシャーレに入れられます。私たちの文化は、UVC光度計を用いて測定される〜1 J/m2/sec、の照射線量率を生成する15ワット殺菌灯からの距離に配置されています。文化は、50 J/m2を照射して、バックにすぐに配置され、実験期間中は37℃のインキュベーターを振る。この投与量は平均、1シクロブタン型ピリミジン二量体ssDNAのすべての4.5キロバイトで、生成します。黄色の照明は、光回復酵素によるシクロブタンのピリミジンダイマーの光回復反転を防ぎます。 </li></ol> 2。 DNAの分離。 <ol><li>複製の中間体が検査される時期に、文化の0.75 mlのアリコートを0.75 mlの氷冷NET30バッファー（100mMのNaCl、10mMのトリス、pH8.0、30mMのEDTA）に入れているとの終わりまで、氷上に置いた時間経過。我々は通常は0で検査試料、15、30、45、60、および90分で、90分の時間経過を実行する。 EDTAと冷たい温度を効果的に複製とヌクレオチド除去修復を停止するのに役立つ。 </li><li>各サンプルは1.5 mg / mlのリゾチームとTEは0.2 mg / mlのRNaseA（10 mMトリス、pH 8.0、1mMのEDTA）150μlの中に再懸濁し、そして20分間37℃で溶解し、その後ペレットです。この時点で、20％のサルコシルのプロテイナーゼK（10mg/ml）と10μlのの10μlを添加し、そのインキュベーションは1時間継続して許可されます。プロテイナーゼKおよびサルコシルは、フェノール抽出が発生する前に、細胞膜やタンパク質に関連するかもしれないDNA断片を解放するのに役立ちます。積極的に複製するDNAは、しばしばタンパク質や細胞膜複合体にバインドされているので、これは回収されているレプリケーションフラグメントの収量を高めることができます。 </li><li>次に、試料を各試料にフェノールの2容量を追加することによって抽出され、チューブを穏やかに5分間反転されています。その後、クロロホルム/イソアミルアルコール（24 / 1）の2容量を加え、このチューブを穏やかに5分間再び反転している。 </li><li>サンプルは、5分間微量遠心機で14,000 rpmで遠心分離し、各サンプルの上部の水相を除去し、新しいチューブに入れ。その後、クロロホルム/イソアミルアルコール（24 / 1）の4倍量を添加し、チューブを穏やかに5分間反転させ、5分間14,000 rpmで再び遠心分離されています。 </li><li>各サンプルの最上位、次いで水相をビーカーに0.1 × TEを250mlに浮かぶ47ミリメートルワットマン0.025μmの細孔、ディスク上の各サンプル100μLをスポッティングして1時間透析する。一本鎖領域または分岐点で複製構造はせん断の影響を受けやすいので、一般的に口を広げてとせん断力を最小限に抑えるためにカミソリの刃で私たちのピペットチップを切断。 DNAを制限酵素で消化されるまで、一般的には、ピペッティングは最小限に抑える必要があります。 </li><li>各サンプルは、複製起点からすぐ下流プラスミドを線形化をPvuII（New England Biolabs）で消化する。 </li><li>前のアガロースゲル、100μlのクロロホルムとブロモフェノールブルーとキシレンシアノールを含む6Xローディング色素の20μlので読み込むために、各サンプルに添加し、混合されています。その後、ローディング色素を含有する水相の40μlのは、ゲルにロードされます。構造的バイアス、構造的なアーティファクト、およびDNAのせん断を最小限に抑えるために、この分離手順では、細胞溶解に続く一本鎖領域、沈殿、またはDNAサンプルを濃縮するために豊かにするすべての手順が含まれていません。 </li></ol> 3。 2Dゲルおよびサザン分析。 <ol><li> 2次元アガロースゲル分析は、3から変更されます。 1次元の場合は、制限されたDNAサンプルが1V/cmで1X TBEは0.4％アガロースゲルを介して実行されています。 10X TBEストック溶液の1リットルは含まれています： <ul><li> 108グラムトリスベース</li><li> 55グラムのホウ酸</li><li> 40ミリリットル0.5M EDTA（pH 8.0）を</li></ul></li><li>ラムダのHind IIIサイズマーカーが最初のレーンにロードされ、その後、サンプルはすべての他のレーンにロードされます。我々は通常、最初のDを実行するimension〜12月15日時間。車線をスキップすることが容易番目の次元にキャストする車線をスライスすることができます。低電圧および低％のアガロースゲルは、主にサイズに基づいてDNA断片を分離するのに役立つ。 </li><li>二次元の場合は、ゲルのレーンは大型肉屋のナイフを使用して、最初の次元ゲルから切り出しています。ラムダのHind IIIマーカーを含む最初の車線は、エチジウムブロマイドで染色することができます。ガイド、作物としてのラムダをHind IIIマーカーを使用し、直鎖状プラスミドの実行を想定している場所の下であるゲルの領域を破棄する。これらの条件下では、我々は、線形化されたpBR322には若干キシレンシアノールの上に汚れを実行すること。見つける</li><li>番目の次元をキャストするには、各レーンは、空のゲルキャスターの上部に水平に配置されます。 1X TBE中の1.0％アガロース溶液を55℃に調製し、そして冷却されるこの時、ゲル溶液が完全にゲルスライスをカバーすることを確認し、番目の次元をキャストするために注がれています。ゲルがセットされると、それはバッファが循環することができる電気泳動装置で6.5V/cmで実行されます。我々は通常、二次元目〜5.5から7時間を実行する。高電圧と高いパーセントアガロースゲルは、効果的にDNAをその形状に基づいてフラグメントだけでなく、大きさを分離。非線形の形状は、二次元目を通して、より実行速度が遅く。 </li><li>以下の電気泳動は、ゲルは、H 2 Oですすぎ、15分間0.25M塩酸の400ミリリットルで2回洗浄し、H 2 Oをリンスしている、、、その後30分間400ミリリットル0.4MのNaOHで2回洗浄。酸は、より効率的に転送する小さな断片に部分的に刻み目にDNA分子を提供洗う。このステップでは、原因で大きなサイズとDNA複製の中間体のユニークな形に必要です。 </li><li>ゲル中のDNAをハイボンドN +ナイロンメンブレン4に転送されます。我々は、0.4M NaOHで下向きのアルカリトランスファーシステムを使用してください。簡単に言えば、0.4M NaOHに浸したブロッティングペーパーの2枚の紙タオルの大規模なスタックに置かれます。ナイロンメンブレンは、0.4M NaOHに浸漬し、吸い取り紙の上に配置されます。次いでゲルを注意深くNaOH溶液で湿らせたブロッティング紙の別の部分、続いて、これの上に階層化されています。最後に、接液吸い取り紙の長い作品は、上部に階層化され、その両端を芯として機能するように0.4M NaOH溶液の1リットルを含む皿に置かれます。我々は通常、6〜12時間のためのDNAの転送をしましょう​​。 </li><li>ナイロン膜が除去され、5 × SSC緩衝液で20〜30秒を洗浄し、そして10.5ミリリットルプレハイブリダイゼーション溶液を含むハイブリダイゼーションのローラーボトルに入れ。その後、メンブレンを42℃以上6時間のための回転と℃でインキュベートする。プレハイブリダイゼーション溶液： <ul><li> 5.0ミリリットルホルムアミド</li><li> 0.5ミリリットル20％SDS </li><li> 2.5ミリリットル20X SSCの* </li><li> 2.0ミリリットル50Xデンハルト* </li><li> 0.5 mlのサケ精子DNA（10mg/ml） SSCおよびデンハート用*レシピは5に記載されています</li></ul></li><li>プレハイブリダイゼーションの期間中、pBR322プラスミド1μgを[32 P] - dCTPで標識したαを使用してロシュから提供されたプロトコルに従ってニックトランスレーションにより32 Pで標識されている。放射性標識プローブ（100μL）●スクリューキャップのマイクロ遠心チューブに10分間98℃インキュベートして変性させ、その後、氷上に即座に配置されます。 </li><li>変性したプローブが含まれている5.9ミリリットルのハイブリダイゼーション溶液に追加されます。 <ul><li> 3.0ミリリットルホルムアミド</li><li> 1.5ミリリットルSSC </li><li> 1.0ミリリットルH 2 O </li><li> 0.15ミリリットル50Xデンハルト</li><li> 0.10ミリリットル20％SDS </li><li> 0.15ミリリットル10mg/mlサケ精子DNA </li></ul></li><li>プレハイブリダイゼーション溶液は、ローラーボトルから注がれており、ハイブリダイゼーション溶液は、ローラーボトルがその後12時間以上42℃のインキュベーターと回転に返されるインチ注がれています。 </li><li>ハイブリダイゼーション溶液は、ローラーボトルから注がれる。次に、ブロットは、回転で0.5X SSC、42℃で0.1％SDS ° Cを含む洗浄溶液の〜150 mlのローラーボトルで20分間4回洗浄される。 </li><li>液体が目に見えて消失するまで最後の洗浄の後、メンブレンをペーパータオル上に配置されます。膜は、その後、ポリ塩化ビニールのプラスチックラップで包み、phosphorimager画面に公開されます。我々はストーム840とその関連ImageQuantソフトウェアを用いて放射能を視覚化し、定量化する。 </li></ol> 4。代表的な結果： <img src="/files/ftp_upload/2220/2220fig1.jpg" alt="図1" /> 図1。プラスミド複製の中間体は、紫外線誘発DNA損傷の存在下および不在下で観察。2D -アガロースゲル分析によって観測されたプラスミドpBR322を消化PvuIIでの移行パターンを図示しています。非複製リニアプラスミドは線形4.4 - kb断片として実行されます。複製プラスミドは、彼らの大規模化、無に起因する非複製のフラグメントよりも遅く移行Y字型構造を形成nlinear形状。この移行のパターンはよく向かって線形領域から伸びる弧を形成している。以下のUV照射、ダブルYまたはX字型の分子は、Y字型の分子の弧よりもゆっくりと移行する円錐領域で観察される。 2DゲルpBR322に32P標識したプローブのサザン解析の例は、すぐにUV照射し、UV照射後15分（出版社から許可を得て複製した）後の細胞のために示されています。

<ol>
	<li>Davis, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Isolation+of+Biochemically+Deficient+Mutants+of+Bacteria+by+Means+of+Penicillin.">The Isolation of Biochemically Deficient Mutants of Bacteria by Means of Penicillin.</a> Proc Natl Acad Sci U S A. 35, 1-10 (1949).</li><li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Damage-Induced+Replication+Fork+Regression+and+Processing+in+Escherichia+coli.">DNA Damage-Induced Replication Fork Regression and Processing in Escherichia coli.</a> Science. 299, 1064-1067 (2003).</li><li>Friedman, K. L., Brewer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+replication+intermediates+by+two-dimensional+agarose+gel+electrophoresis.">Analysis of replication intermediates by two-dimensional agarose gel electrophoresis.</a> Methods Enzymol. 262, 613-627 (1995).</li><li>Spivak, G., Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+damage+and+repair+in+specific+DNA+sequences.">Determination of damage and repair in specific DNA sequences.</a> METHODS: A Companion to Methods in Enzymology. 7, 147-161 (1995).</li><li>Sambrook, J., Russell, D. W. <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 Cloning - A laboratory manual. , (2001).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvABC+is+required+to+resolve+holliday+junctions+that+accumulate+following+replication+on+damaged+templates+in+Escherichia+coli.">RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli.</a> J Biol Chem. 281, 28811-28821 (2006).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvAB+and+RecG+Are+Not+Essential+for+the+Recovery+of+DNA+Synthesis+Following+UV-Induced+DNA+Damage+in+Escherichia+coli.">RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli.</a> Genetics. 166, 1631-1640 (2004).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecO+Acts+with+RecF+and+RecR+to+Protect+and+Maintain+Replication+Forks+Blocked+by+UV-induced+DNA+Damage+in+Escherichia+coli.">RecO Acts with RecF and RecR to Protect and Maintain Replication Forks Blocked by UV-induced DNA Damage in Escherichia coli.</a> J Biol Chem. 279, 3492-3496 (2004).</li><li>Belle, J. J., Casey, A., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+DnaB+helicase+leads+to+the+collapse+and+degradation+of+the+replication+fork:+a+comparison+to+UV-induced+arrest.">Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.</a> J Bacteriol. 189, 5452-5462 (2007).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecBCD+and+RecJ/RecQ+initiate+DNA+degradation+on+distinct+substrates+in+UV-irradiated+Escherichia+coli.">RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli.</a> Radiat Res. 168, 499-506 (2007).</li><li>Al-Hadid, Q., Ona, K., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecA433+cells+are+defective+in+recF-mediated+processing+of+disrupted+replication+forks+but+retain+recBCD-mediated+functions.">RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions.</a> Mutat Res. 645, 19-26 (2008).</li></ol>

利害の衝突は宣言されません。

紫外線による損傷の存在下および非存在下で野生型の細胞から得られた典型的な結果を図1に示されています。細胞が急速に指数増殖期で成長しているときに損傷がない場合には、合計プラスミドDNAの〜1％がYの円弧で見つけることができます。ブロックされた複製フォークが損傷部位に蓄積するように照射後、Y字型の分子の一過性増加が観察される。 X字型の複製中間体はまた、一時的に蓄?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>An example of the Southern analysis of the 2D gel probed</td>
<td>&nbsp;</td>
<td>GE</td>
<td>G15T8</td>
<td>Yellow Lighting</td>
</tr>
<tr>
<td>15 &mu;watt germicidal lamp</td>
<td>&nbsp;</td>
<td>Sylvania</td>
<td>F20T12/GO</td>
<td>UV Lamp</td>
</tr>
<tr>
<td>Blak-Ray UV Intensity Meter 254nm</td>
<td>&nbsp;</td>
<td>Daigger</td>
<td>EF28195T</td>
<td>UVC photometer</td>
</tr>
<tr>
<td>0.025 &mu;m pore disks</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>VSWP04700</td>
<td>Floating dialysis disks</td>
</tr>
<tr>
<td>PvuII</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>ER0632</td>
<td>Restriction Endonuclease</td>
</tr>
<tr>
<td>Nick-translation kit</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>976776</td>
<td>To make 32P-labeled probe</td>
</tr>
<tr>
<td>Blotting Paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-704</td>
<td>For Southern transfer</td>
</tr>
<tr>
<td>Nylon membrane</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>RPN203S</td>
<td>For Southern transfer</td>
</tr>		</tbody>
		</table>
		</div>

visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

DNA損傷の存在下で不正確な複製は、すべての細胞型で観察し、広く直接ヒトの癌の開発に関連付けられていると信じられている細胞の再配列と特異的変異の大部分を担当しています。このような紫外線照射によって誘導されるようなDNA損傷は、著しく正確にゲノムテンプレートを複製する複製の能力を損なう。遺伝子産物の数は、複製が鋳型にDNA損傷を検出すると、必要とされるが確認されている。しかし、残りの課題は、これらのタンパク質は、複製中に病変の処理方法を決定することでした<em&gt; in vivoで</em&gt;。モデル系として大腸菌を用いて、我々は、2次元アガロースゲル分析がreplicatingプラスミドに生じた構造的な中間体を識別するために使用できる手順を説明します。<em&gt; in vivoで</em&gt;以下の紫外線誘発DNA損傷。この手順は、紫外線による損傷によってブロックされた複製フォークは、RecAタンパク質とRecFの経路に関連付けられているいくつかの遺伝子産物によって安定化される一時的な反転を受けることを示すために使用されています。技術は、これらの複製の中間体は、ヌクレオチド除去修復と複製が再開による病変の除去と相関する時間まで維持されていることを示している。

Watch this Scientific Journal Video about Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis at JoVE.com

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

我々は、2次元アガロースゲル分析は、UV照射後に発生する複製の中間体の構造を識別するために使用できるようにするための手順を示します。

のUV -誘発複製中間体の可視化 E.大腸菌二次元アガロースゲル分析を用いた

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

visualization-uv-induced-replication-intermediates-e-coli-using-two

Research

JoVE Journal

Biology

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

10.6K ビュー.  Portland State University. 我々は、2次元アガロースゲル分析は、UV照射後に発生する複製の中間体の構造を識別するために使用できるようにするための手順を示します。

ビデオ: Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

ヒートショック法による大腸菌へのプラスミドDNAの形質転換

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

生物学

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

アガロースゲルベースの​​マイクロ流体デバイスを用いた神経幹細胞運動の評価

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

セミ変性洗剤-アガロースゲル電気泳動によりアミロイド凝集のためにスクリーニング

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

内タンパク質の定方向進化のための変異導入と機能選択プロトコル E.大腸菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

DNAの繊維技術を用いた脊椎動物のモデルシステムのDT40のDNA複製の可視化

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

DNA断片の分離のためのアガロースゲル電気泳動

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

ターゲティングされたのsRNAのファージ媒介送達はにおける遺伝子発現をノックダウンするために構築します<em&gt; E。大腸菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

ヒトノロウイルス突出ドメインの生産で<em&gt; E。大腸菌</em&gt; X線結晶用

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

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 ...