Name: streamlined purification of plasmid dna from prokaryotic cultures
Uploaded: 2011-01-05T17:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

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。

<table><tbody><tr><td>AcroPrep Advance Filter plate</td><td>Pall Corporation</td><td>PN 8175 (1 mL) or PN 8075 (350 &amp;#181;l)</td><td>3 &amp;#x03BC;m glass fiber 0.2 &amp;#x03BC;m Supor&amp;#174; membrane for clarification</td></tr><tr><td>AcroPrep Advance Filter Plate</td><td>Pall Corporation</td><td>PN 8132</td><td>for DNA Binding</td></tr><tr><td>Vacuum manifold</td><td>Pall Corporation</td><td>PN 5017</td><td></td></tr><tr><td>Vacuum pressure pump</td><td>Pall Corporation</td><td>PN 13157</td><td></td></tr></tbody></table>

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

19.9K ビュー.  Pall Life Sciences.  中文 

ビデオ: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

酵素的に活性G4 Resolvase1の精製のためのG-四重鎖DNA親和性のアプローチ

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

生化学

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

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

浄化、体外(p) ppGppクロストリジウム ・ ディフィシルから合成酵素の活性

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

免疫・感染学

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

アフィニティータグ組換えタンパク質のハイスループット精製

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

培養哺乳動物細胞での超並列レポーターアッセイ

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow&#39;s ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method&#39;s true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate counting.

オープンソースの液滴マイクロ流体工学によるプラスミド安定性解析

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating ...

生物工学

Use of In Vivo Assembly for High-efficiency Plasmid Construction

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA fragments to assemble plasmids. This method functions by transforming DNA fragments with regions of 15-50 bp of homology into commonly used laboratory Escherichia coli strains and the bacteria use the RecA-independent repair pathway to assemble the DNA fragments into a plasmid. This method is more rapid and cost-effective than many molecular cloning methods that rely on in vitro assembly of plasmids prior to transformation into E. coli strains. This is because in vitro methods require the purchase of specialized enzymes and the performance of sequential enzymatic reactions that require incubations. However, unlike in vitro methods, IVA has not been experimentally shown to assemble linear plasmids. Here we share the IVA protocol used by our laboratory to rapidly assemble plasmids and subclone DNA fragments between plasmids with different origins of replication and antibiotic resistance markers.

In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.

高効率プラスミド構築のための In vivo アセンブリの利用

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA ...

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 ...

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

CAPRRESI：プラスミド回収および制限酵素部位挿入によるキメラアセンブリ

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...

原核生物培養液からプラスミドDNAの合理化精製

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酵素的に活性G4 Resolvase1の精製のためのG-四重鎖DNA親和性のアプローチ

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

浄化、体外(p) ppGppクロストリジウム・ディフィシルから合成酵素の活性

アフィニティータグ組換えタンパク質のハイスループット精製

培養哺乳動物細胞での超並列レポーターアッセイ

オープンソースの液滴マイクロ流体工学によるプラスミド安定性解析

高効率プラスミド構築のための In vivo アセンブリの利用

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

真核細胞から体外の環状DNAのゲノムワイドな精製

CAPRRESI：プラスミド回収および制限酵素部位挿入によるキメラアセンブリ

原核生物培養液からプラスミドDNAの合理化精製

文字起こし

要約

さらに動画を探す

この動画の章

酵素的に活性G4 Resolvase1の精製のためのG-四重鎖DNA親和性のアプローチ

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

浄化、体外(p) ppGppクロストリジウム ・ ディフィシルから合成酵素の活性

アフィニティータグ組換えタンパク質のハイスループット精製

培養哺乳動物細胞での超並列レポーターアッセイ

オープンソースの液滴マイクロ流体工学によるプラスミド安定性解析

高効率プラスミド構築のための In vivo アセンブリの利用

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

真核細胞から体外の環状DNAのゲノムワイドな精製

CAPRRESI：プラスミド回収および制限酵素部位挿入によるキメラアセンブリ

浄化、体外(p) ppGppクロストリジウム・ディフィシルから合成酵素の活性