1。ライセートクリアランス<ol><li> E.を拡大37℃で一晩、適切な抗生物質とルリアブロスのディープウェルプレートにおける所望のプラスミドDNA（文化/ウェルを1mL）°振とう℃で形質転換した大腸菌の培養。 </li><li>その後、10分間5000 × gでの培養プレートでペレット大腸菌 、上清をデカント。 </li><li> 100μL再懸濁バッファー（50mMのTris - HCl、pH8.0の、10mMのEDTA、100μg/ mlのRNaseの）内の各ペレットを再懸濁する。 </li><li> 100μL/ウェル溶解バッファー（200 mMのNaOHを、1％SDS）を加え、2分間プレートシェーカーを用いて振る。 </li><li> 100 /ウェル中和バッファー（3.0 M酢酸カリウム、pH5.5）に追加し、2分間振とうする。 </li><li>説明プレートをライセートに細胞ライセートを転送する。 </li><li>真空マニホールドの底面に350μLコレクションプレートと場所の上にDNA結合プレートを置きます。 （図1を参照）真空マニホールドを組み立てます。 </li><li>真空装置の上にライセートの清澄化プレートを置きます。 </li><li>水銀10インチ（0.34 bar）で真空状態にしてDNA結合プレートにろ液を集める。水銀12インチよりも大きい真空を使用しないでください。 （0.41バール）。 </li><li>吸引マニホールドを分解します。装置の底部に2mLの廃棄物収集プレートを置きます。 </li></ol> 2。 DNA結合<ol><li>マニホールドの上にDNA結合プレートを移動する（図2を参照）。 </li><li> 300μL/ウェル結合バッファー（6 Mグアニジン- HCl）を追加し、混在させると上下にピペッティング。 </li><li> [遅い真空用水銀インチ〜5（0.17バール）]真空を適用し、ろ液を捨てる。 DNAが今膜に結合しています。 </li><li>洗浄緩衝液（80％エタノール）の400μL/ウェルで洗ってください。 </li><li>真空を適用し、ろ液を捨てる。 2 mlのコレクションプレートを使用したり、無駄に直接フィルタする場合、不要な捨てる。 </li><li>一度洗浄を繰り返します。 </li><li>乾燥して吸収性タオルにフィルタープレートの底にしみ。 </li><li>残留アルコールの除去を確実にするために5〜10分に一回以上の真空適用されます。 </li><li>エタノール液滴の除去を確実にするために底部を覆い隠す。 </li></ol> 3。 DNAの溶出<ol><li> 70ウェル/μlの溶出バッファー（10mMトリス- HCl、pH8.0の、1mMのEDTA）を追加します。高いDNA濃度が所望される場合、ボリュームは50μL/ウェルに低減することができるが、平均総収率が低くなります。 </li><li> 1分間室温でインキュベートする。 </li><li>精製したDNAは、真空または遠心分離のいずれかで溶出させることができる。 </li><li>真空法：真空マニホールドに清潔なコレクションプレート（DNase処理、RNaseフリー）。すべての溶出緩衝液は、DNA結合プレートを通過するまで吸引マニホールドの上にフィルタープレートを置き、1分間はHg（0.5バール）15インチの真空を適用する。 （図3を参照）DNAを精製収集する。 </li><li>遠心法：5分間1,000 × gでクリーンなコレ​​クションプレートや遠心の上に配置浄化フィルタープレート。 </li></ol> 4。定量的および定性的分析<ol><li> OD 280分の260を測定し、DNAを精製したの濃度を計算する。 </li><li>精製PDNAのアガロースゲル分析を行います。 </li></ol> 5。代表的な結果 大腸菌は、従来の遠心分離とポールアクロアドバンスライセートの清澄化フィルタープレートを使用して明らかにライセートを出発原料から破片のかなりの減少を示した。 （図4）。 3つの濃度でのプラスミドDNAをTEバッファーに添加し、アクロアドバンスライセートの清澄化フィルタープレートを通過させた。プラスミドDNA濃度の比較は、事前事後のろ過、ショーは3つのすべての濃度で100％の回収（図5）を見る。 明らかE.からのDNAの回収を実証するために、 大腸菌ライセート、プラスミドDNAは、粗溶解物に添加し、濾過によって清澄化した。同じプラスミドDNAは、ろ過後の細胞のライセートに添加した。電気泳動は、各サンプルからアリコート（図6）上で行った。各サンプルから同様の強度の単一の、明確なバンドは、ろ過ベースの明確化のステップの間に少しあればDNAの損失を示しています。 E.の文化PCATプラスミドDNAを含む大腸菌は、大腸菌から精製した大腸菌は、ポールの350μL及び1mLのDNA結合フィルタープレートだけでなく、350μLDNA結合プレートの二つのブランドを用いてライセート。メーカーの推奨するプロトコルで指定されたすべての精製を行った。最高DNA濃度と総収量は、ポールの1 mLのアクロプレップアドバンスDNA精製フィルタープレート（図7および表2）から見られている。 他社Wも高いDNA濃度を（155 ng /μLの）示していますが、全DNAの収量は、（4.7μgの/ウェル）低い回復量に起因する最も低いです、〜30μL。競合他社のMは、5.6μgの/ウェルの総収率で108 ng /μLのでDNAの最低濃度を与えた。 寒天つの異なるDNA結合プレートからPCAT DNAを精製したのOSEゲル分析は、図8に示されています。各収集されたサンプルの量は、電気泳動の前に回復量の差を補正するために65μLに調整した。すべてのサンプルは、スーパーコイルプラスミドDNAを示しているが、フィルタープレートの各々からのDNA収量が異なります。 図9に調製されたDNAはスーパーコイル構造を取ってすべてであると同様の濃度が表示されます。 <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="図1" /> 図1。ライセート清澄化のための真空マニホールドの組み立て 。 <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="図2" /> 図2。 DNA結合のための真空マニホールドの組み立て 。 <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="図3" /> 図3。真空溶出用真空マニホールドの組み立て 。 <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="図4" /> 図4。ろ過は、サンプルの説明を簡素化します 。吸引ろ過（350μLアクロプレップアドバンスライセートの清澄化フィルタープレート）または従来の遠心分離によって清澄化粗溶解物の300μLのトリプリケートサンプル。 OD 320は、明確化の前と後に測定。 <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="図5" /> 図5。アクロアドバンスライセートの清澄化フィルタープレート通過後PDNAの完全回復 。 300μLのTEバッファーには25、50および100μg/ mLでPCATでスパイク。 DNA濃度と回収率は、N = 2の前とろ過後のOD 260から計算。 <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="図6" /> 図6。説明後のPCAT DNAの回収 。その後、明確化の前または後のライセートに添加pUC19の同量、TEバッファーで10に1を希釈した。 1.2％アガロースゲル上の2μL/レーンにロード。 <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="図7" /> 図7。競合他社のプレートよりも飽きが来るアクロアドバンスDNA結合フィルタープレートでウェルあたりの高いプラスミドDNAの収量。1mLの（ポールと競合M）または1.5 mLで表示されたDNAの精製プレートを使用してPCATプラスミドDNAの収量（OD 260）（他社W）の一晩培養DH5α。板の製造業者の推奨するプロトコルを使用して精製。エラーバーは標準誤差（n個&gt; 6）を示している。 <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="図8" /> 図8。 PCATのDNAのアガロースゲル分析では、指示されたDNA結合プレートで精製 。精製PDNAの1.2％アガロースゲル電気泳動。 3つの別々の精製からプールされたサンプルは、溶出液は、精製、1:10に希釈した後、65μLに調整。レーンあたり2μLをロード。 <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="図9" /> 図9。遠心または吸引方法は精製PDNAの最終的な溶出液を収集アガロースゲル電気泳動に使用されているときは同じような純粋なDNAの質と回復 。アクロプレップアドバンスフィルタープレートの製造業者の推奨するプロトコルは、PDNAの精製のために続いた。溶出は、2分間はHg（0.5バール）15インチで5分間または真空のために1,000 × gで遠心することにより行った。

このビデオ記事の著者は、ビデオとテキストで紹介さ測定器と試薬を生成ポールライフサイエンスの従業員です。

我々は、いくつかの利点に支払うアクロアドバンスフィルタープレートの安易な、合理化されたプロトコルを記述する。手動、半自動、また​​は完全に自動化されたアクロアドバンスの形式で処理したときにこのメソッドは、最大プラスミドDNAの収量が用意されています。ライセートクリアランスプレート上で統合されたプリフィルタは、偶数サンプル、微粒子のものを含む高いレベルの一貫性のあるろ過を提供します。より速く、より均一な最小限のホールドアップ量とプレート間の濾過速度のプレートの結果のほかジオメトリ、およびコンセントの先端の形状は、クロスコンタミネーションの心配なく、レシーバープレートへの試料の直接の流れを提供します。重要なのは、アクロアドバンスフィルタープレートのDNA結合プレートに高い結合容量膜は、高品質のプラスミドDNAの最大の回収用に最適化されています。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>AcroPrep Advance Filter plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8175 (1 mL) or PN 8075 (350 &mu;l)</td>
<td>3 &mu;m glass fiber 0.2 &mu;m Supor&reg; membrane for clarification</td>
</tr>
<tr>
<td>AcroPrep Advance Filter Plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8132</td>
<td>for DNA Binding</td>
</tr>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 5017</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pressure pump</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 13157</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

streamlined purification of plasmid dna from prokaryotic cultures

我々は、原核生物の培養液から開始し、高純度のDNAで終わる、96プラスミド調製のためのアクロアドバンスフィルタープレートの完全なプロセスを説明します。細菌溶解物のクリアランスとDNA精製のためのマルチウェルろ過に基づいて、この方法では、プラスミド調製のための合理的なプロセスを作成します。シリカベースのメ​​ディアを含むフィルタープレートを簡単に真空ろ過やプラスミドDNAのかなりの量を得るために遠心分離で処理することができます。定量分析は、精製されたプラスミドDNAは、1.97の平均OD 280分の260の比率で高品質の一貫しているかを判断。全体的に、プラスミド収量は、シークエンシングやクローニングなどの下流のアプリケーションに、より純粋なDNAを提供する。アクロアドバンスフィルタープレートを使用してのこの合理化された方法は手動、半自動または全自動処理が可能になります。

Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

このプロトコルは、より効率的な並列明確化し、プラスミドDNA精製のための費用対効果の高い代替手段です<em&gt; E.大腸菌</em&gt;文化。アクロアドバンスプロセスは、高い結合容量のDNA結合フィルタープレート上で精製して最適化されたライセートの清澄化フィルタープレートから始まります。

原核生物培養液からプラスミドDNAの合理化精製

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

streamlined-purification-of-plasmid-dna-from-prokaryotic-cultures

Research

JoVE Journal

Biology

19.8K ビュー.  Pall Life Sciences.  中文 

ビデオ: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

生物学

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 ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

骨格筋からの前駆体の精製

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

キネシンの単離及び精製からショウジョウバエ胚

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

前のDNA精製せずに植物や動物のサンプルの遺伝子型

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CASシステムの酵素のための基質の生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

実装化学反応ネットワーク用プラスミド由来のDNA鎖置換ゲイツ

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

真核細胞から体外の環状DNAのゲノムワイドな精製

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...