1。 DNAの定量のための手順<ol><li>楽器の電源をオンにします。 </li><li> PCのデスクトップからBioSpec - nanoソフトウェアを開きます。 </li><li>シンプルな核酸二本鎖DNAのタブ（図1）をクリックしてください。 </li><li>ソフトウェアは、計測器との通信を確立し、初期化シーケンスを実行します。これは、測定器が仕様に従って実行していることを確認します。 </li><li>光路長スライダーで0.7または0.2 mmを選択します。これは、光路長を選択し、すべての計算は、選択された光路長に基づいています。 </li><li> BioSpec - nanoソフトウェアをクリックしてセットアップ- Autowipingのセットアップの次の。 1または2を選択します。 </li><li>ピペット2μL（0.7 mm用）、H 2 Oまたは台座上にバッファ。 </li><li>ブランク測定用の空白またはF6をクリックします。新しい光路長が選択されるたびに、それは空白を記録する必要があります。 </li><li>台座上のサンプルのピペット2μL。 </li><li>ソフトウェアのスタートや本体のスタートボタンを押してクリック。アッパーウィンドウには、サンプル液滴と接触するために移動します。キセノン​​フラッシュは観察することができます。 </li><li>再現性を調べるには、同じサンプルを3回繰り返します。 </li><li>記録されたすべてのデータが。芽のファイルとして保存されます。 </li><li> 。pdfまたは。csvファイルとして保存するには、PDF / CSVまたはF9を保存]をクリックします。結果は、[保存]タブで適切な選択を使用して。csvファイルとして保存することができます。 </li></ol> 2。タンパク質定量のためのプロトコル<ol><li>タンパク質定量のタブを選択します（図2）。 </li><li>既知の場合、吸光係数のE280を入力してください。 </li><li> E280が知られていない場合、それはe280Calcを使用して計算する。 </li><li>光路長スライダーで0.7または0.2 mmを選択します。 </li><li>セットアップ- Autowipingのセットアップ]をクリックします。 2を選択するか、上記の。 </li><li>ピペット3μL（0.2 mm用）、H 2 Oまたは台座上にバッファ。 </li><li>ブランク測定用の空白またはF6をクリックします。 </li><li>台座上のタンパク質サンプルをピペット3μL。 </li><li>ソフトウェアのスタートや本体のスタートボタンを押してクリック。 </li><li>再現性を調べるには、同じサンプルを3回繰り返します。 </li><li>記録されたすべてのデータが。芽のファイルとして保存されます</li><li> 。pdfまたは。csvファイルとして保存するには、PDF / CSVまたはF9を保存]をクリックします。 </li></ol> 3。代表的な結果： 1。 DNAの定量の結果図3は、典型的な二本鎖DNAのサンプルの測定からデータを示しています。濃度と計算されたOD 280分の260比も表示されます。図4は、DNAの定量のための再現性のテスト結果が含まれています。図3と図4に示す結果のデータは、0.26％と低く、相対標準偏差から明らかなように、再現性の高いです。 注1： DNAの定量が不正確には紫外領域で吸収することができるバッファー成分の存在から生じる可能性があります。また、均質な試料溶液を使用する必要があります。ピペッティングされているサンプルの量はわずか1〜2μLであるとして、あらゆる気泡の導入なしに正確な量を注意深くピペット操作が必要です。 2。タンパク質定量の結果図5は、BSAのためのタンパク質濃度の分析を表します。 μg/ mLのタンパク質濃度は、E280の値に基づいています。図6は、再現性の分析を示しています。 DNAのためだけでなく、タンパク質のためだけではなく - それはBioSpec -ナノは正確に濃度を測定することが0.51％、相対標準偏差から明らかである。 注2：タンパク質の濃度に影響を与える要因 タンパク質の正確な濃度測定は、pH、緩衝液、温度、または添加物に起因する不安定性から最小の影響​​がある場合に有効です。最も精製されたタンパク質は、処理の様々なステップを通過します、従ってそれは、タンパク質が液滴の形成を妨げる可能性界面活性剤または他の添加剤を含んでいる可能性があります。そのようなサンプルについては、5 mmの光路長の石英セルを使用することを強くお勧めします。 <img src="/files/ftp_upload/2699/2699fig1.jpg" alt="図1" /> 二本鎖DNAの定量化のため図1の分析物の選択ウィンドウ<img src="/files/ftp_upload/2699/2699fig2.jpg" alt="図2" /> 非標識タンパク質の定量については、 図2の分析物の選択ウィンドウ<img src="/files/ftp_upload/2699/2699fig3.jpg" alt="図3" /> DNAの定量化のための図3。代表データ<img src="/files/ftp_upload/2699/2699fig4.jpg" alt="図4" /> DNAの定量については、 図4。再現性のデータ<img src="/files/ftp_upload/2699/2699fig5.jpg" alt="図5" /> 図5。タンパク質定量のための代表的なデータ<img src="/files/ftp_upload/2699/2699fig6.jpg"altが=&quot;図6&quot;/&gt; 図6。タンパク質定量のための再現性のデータ

<ol>
	<li>Aisubel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Short Protocols in Molecular Biology. , (1999).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+and+predict+molar+absorption+coefficient+of+a+protein.">How to measure and predict molar absorption coefficient of a protein.</a> Protein Science. 4, 2411-2423 .</li></ol>

Suja Sukumaran、博士はこの記事で紹介楽器を作る島津の従業員です。

BioSpec - nanoはこのような矛盾した結果と面倒な肉体労働のようなDNAの定量方法に固有の現在の問題を、解決するために設計されています。各測定値は、経験豊富な分子生物学者が1分間に3から6についての複数のサンプルを分析できるように、わずか3秒かかります。ユニークなワイパー機能は、測定のスループットを向上するだけでなく、測定値の間に無視できるキャリーオーバーで、その結果、台座の手動クリーニングが不要となります。さらに、1000 ng /μLのを含むDNAサンプルのためわずか2ワイプを使用してテストでは、キャリーオーバーを示した2未満ng /μLの。 5 mmの光路長のセルの可用性は、希薄な核酸や蛋白質のサンプルを分析するためのオプションを提供し、他のアプリケーションのための本器の利用を拡張します。

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concentration determination of nucleic acids and proteins using the micro-volume bio-spec nano spectrophotometer

核酸の定量の手順は、最後の三十年で大きく進歩している。ますます、分子生物学者は、彼らの実験のための核酸サンプルの一貫性のある小容量の分析を必要とする。 BioSpec - nanoは特殊な光学系と敏感なPDA検出器を経由して現在のDNAの定量方法に固有の不正確な、非再現性の結果、、の問題に対して可能​​性のあるソリューションを提供します。 BioSpec - nanoはまた、それによって光ファイバー素子と手動洗浄の、退屈で反復的な、そして一貫性のない配置を排除し、取り付け、測定、および洗浄が楽器によって行われるように自動化機能を備えています。 本研究では、データはDNAとタンパク質の定量について、同様の測定再現性と精度で表示されます。自動化されたサンプルの連絡先と迅速なスキャンは、優れたスループット、その結果、3秒で測定することができます。データ解析は、ソフトウェアの組み込み機能を用いて行われる。 DNA濃度を計算するために使用される式は次のとおりです。 サンプルの濃度= DF ·（OD260 - OD320）·農協（1） =サンプルの希釈率とNACF =核酸の濃縮係数をどこDF。 核酸濃度の係数は1を選択した分析物にしたがって設定されています。 タンパク質濃度の結果は、それぞれE280と分子量の値を入力して、μg/ mLのようまたはモル/ Lで表すことができます。チロシン、トリプトファン、システイン（SS結合）のための残留値はe280Calcタブで入力されると、吸光係数の値は、E280のように計算されている= 5500 ×（トリプトファン残基）+ 1490 ×（Tyr残基）+ 125 ×（システインSS結合） 。 E280値は、濃度計算のためのソフトウェアで使用されます。 核酸とタンパク質の濃度測定に加えて、BioSpec - nanoは、他の多くの分析対象物の超マイクロボリューム分光光度計として、あるいは5 mm光路長セルを使用して標準的な分光光度計として使用することができます。

Watch this Scientific Journal Video about Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer at JoVE.com

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

この通信は、dsDNAとタンパク質定量用BioSpec -ナノUV - VIS分光光度計の精度と再現性に関するデータを提示。自動拭き取り機能は、より正確な結果を得るために最小のキャリーオーバーのスループットと結果を改善しながらであっても、超小型のボリューム（1〜2リットル）で、再現性は、優れています。

マイクロボリュームバイオスペックナノ分光光度計を用いて核酸およびタンパク質の濃度測定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

concentration-determination-nucleic-acids-proteins-using-micro-volume

Research

JoVE Journal

Biology

38.9K ビュー.  Scientific Instruments. この通信は、dsDNAとタンパク質定量用BioSpec -ナノUV - VIS分光光度計の精度と再現性に関するデータを提示。自動拭き取り機能は、より正確な結果を得るために最小のキャリーオーバーのスループットと結果を改善しながらであっても、超小型のボリューム（1〜2リットル）で、再現性は、優れています。

ビデオ: Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

光度計2000C分光光度計を使った微量タンパク質の濃度測定

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

速度論と構造機能解析のアフィニティ定量のための適切なタンパク質濃度の重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

日立F - 7000蛍光分光光度計とPicoGreen色素を使用してdsDNAの定量化

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

核酸の光度計微量定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

3DNAを有する核酸構造の分析と構築

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

メチルトランスフェラーゼと補因子類似体核酸およびタンパク質の配列特異的標識化

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

使用モデルタンパク質への非標準アミノ酸の残基特異的取り込み<em&gt;エシェリヒア・コリ</em&gt;無細胞転写翻訳システム

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

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

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

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

核酸の濾過単離：簡易迅速DNA抽出方法

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

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

野生型および肝臓の肝臓試料および牛乳試料中のシアル酸の定量<em&gt; CMAH</em&gt;ノックアウトマウス。

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...