我々は標本を得るための支援のためにアランタスカー（USDA-APHIS、リバーデール、MD）、スティーブン·コンプトン（クレムソン）、シェリーAultman（クレムソン）、クレイグ·ラムゼイ（USDA-APHIS、フォートコリンズ、コロラド州）、そしてベティMarose（UMD）に感謝。我々は、ビデオの撮影の彼の仕事のための学生アンドリュー·エイドリアン（UA-ハンツビル）とデレク·タッカー（UA-ハンツビル）は、このプロトコルをテストする彼らの支援のために、ヨセフHerdyに感謝します。この作品は、国立魚類野生生物財団によって資金を供給された。

<ol>
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我々は、開示することは何もありません。

<html>アメリカの保育園や景観産業はエキゾチックで斬新な植物種を栽培し、販売に成功する。貿易の拡大、グローバル化と相まって、これは、侵襲的な植物種は、次のコマンドを入力し確立し、米国では連邦政府の侵襲になる可能性を含めて、頻繁に入手できない情報に依存しているような植物を調節する能力を広がっていく可能性が高くなり、ネイティブと帰化した分類群から正しい分類、遺伝的明瞭。侵略的な植物の知識はしばしば制限されているため、隠された侵襲的な特性を持つインポートされた植物は喜んで彼らだけが私たちの農業と天然資源に侵入することが後で学ぶために導入されました。このプロトコルは、I.に関連付けられているような問題に対処することを目指して正確には、野生型cogongrassとその装飾JBGの相手の元に戻す形を ​​区別できる最初の簡略化の分子のメソッドを提供することによって、 チガヤの品種。 このプロトコルの開発のためjove_content &quot;&gt;は、野生型cogongrassは、2008年6月にジェイ、フロリダ州の近くにサンタローザ郡の池·クリーク森林単位で帰化した集団から採取した。JBGは、商業苗床（ブルーバード保育園、（株）から調達されました。リバー、マサチューセッツ州ユニバーシティパークから2009年6月;）2008年6月に同様にコロンビア州の住宅所有者のコレクションから、MO JBGは2008年6月にクレムソン大学、サウスカロライナ州でキャンベル地質標本館の中庭から得られたが戻り、 2009年にコロンビア、ミズーリにある住宅（リーランドCsekeで識別される）の前庭からすべての工場はハンツビルのアラバマ大学（ハンツヴィル、アラバマ州）に位置する温室で維持した。 これらの植物から採取したDNAの遺伝子配列は、一般的にバーコードの植物2品の独立したDNA領域の深さの比較に含まれています。すべてのケースでは、JBGの配列はこのように助け、JBG戻すのものと100％一致したJBGは確かに緑、侵襲的な形に戻すにはないことを確認してください。唯一の核と葉緑体trnL-Fの領域は、遺伝的にcogongrassとJBGを区別するために使用できる違いがあります。 trnL-Fの領域は、2個のSNPと2 indelsの（挿入および欠失）がありながら、ITS領域は、cogongrassとJBGの間に3個のSNP（一塩基多型）の合計を持っています。これらの遺伝的差異は、野生型cogongrassとJBG戻りを区別することができます開発される様々な特異的PCRプライマーを可能にした。最も信頼性の高い結果がプラスチドtrnL-Fの領域由来のプライマーから来ている。したがって、このプロトコルはcogongrass、JBGの葉緑体ゲノムのtrnL-F領域とJBG（ 図4）元に戻すとの間の配列の違いに基づいています。 問題の種類により具体的なプライマーを生成し、密接に関連する種から偽陽性を避けるために助けるために、アルI. lの知られているtrnL-Fのシーケンスチガヤの品種は、関連する草種（29種から43の独立した配列、 例えば、Cymbopogon citratus、Sorghastrum incompletum、ハトムギハトムギ-jobi、ススキ、サトウキビ、モロコシ ）からtrnL-Fの配列と比較した。 、我々は草、様々な特異的プライマーの特異性の29種を越えて様々な特異的プライマーの配列を調べたが、総合的に他のほとんどの草種からのDNAを増幅する能力について検討されていない。その結果、DNA抽出に使用する組織は、慎重にIとして識別する必要があります前にこのプロトコルを開始するチガヤ 。草がcogongrassまたはJBGのいずれかとして識別できない場合は、我々は、配列がcogongrassまたはJBGと正確に一致することを確認するためにPCR産物をシークエンシングをお勧めします。現在、指定された草の標本の身元を確認するための最も正確な方法は 、tの両方で、PCRを実行することですRNL-FおよびITS領域、配列のPCR産物の確認と正確に識別分類から知られている配列への配列の比較が続く。 DNAは、コントロールは、このプロトコルに詳述プライマー（trnL-Fの領域の場合）または他の出版物13から15で利用可能な他のプライマーを用いて増幅することができます。シーケンスは、私たちの簡略化の手順を使用してより集中的に多くの労力とコストである。 PCRに用いるプライマーの品質は、プロシージャの成功に不可欠です。我々は斜バイオ株式会社（からこの手順のためのプライマーが利用可能になった<a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a> 、ハンツヴィル、アラバマ州）。斜めバイオからプライマーを注文する利点は、彼らが我々の研究室での最適化に用いたプライマーと同一のサンプルの生産シリーズから、各プライマーのアリコートを大量に格納することです。その結果、プライマーは同じ配列を持っていますが、トンだけでなく、ねえ、このプロトコルで使用されたまったく同じ製造ロットから来ています。同じロットからプライマーを用いて、PCRプライマーの品質の違いから生じるの手順で、余分な変数を避けるのを助けることができます。同様に、他のTaqポリメラーゼはPCR、使用されるTaqポリメラーゼの品質のために正常に動作する必要がありますが、PCRの結果の品質に影響を与えます。 PCR試薬の優れた一貫性を可能にするために、我々は、クロンテックからの試薬を用いてプロトコルを最適化している。アドバンテージ2ポリメラーゼ（クロンテック社、カリフォルニア州マウンテンビュー、カタログ番号639201または639202）は、堅牢なホットスタートTaqポリメラーゼと高い特異性とより正確な増幅を提供するのに役立ちプルーフリーディング酵素の混合物である。 このプロトコルは母性草に継承されている葉緑体DNA、に依存しているため、cogongrassとJBG遺伝子型間のハイブリダイゼーションのイベントは私たちの分子同定の手順でキャプチャすることはできません。 hypridizationがsuspeである場合にCTED、我々は両方の親から継承された核領域を使用することをお勧めします。植物ジェノタイピングする際に考慮すべき最も一般的に使用される非プラスチド可変領域は、核リボソームのITS領域13-15,17です。現在、我々は同じPCRチューブ内での核ITS領域のそれと葉緑体trnL-F領域の増幅を多重化に向けて前進している。核DNA領域とプラスチド多重化は、潜在的に単独を使用する場合の制限を回避するだろうが、このような方法はケースバイケースで可能性を判断するために最適化し、追加評価が必要になります。定量的リアルタイムPCR（定量PCR）、およびそのような分子ビーコン（蛍光プライマープローブ）、などの新しい技術の使用は、フェイルセーフの正確な植物ジェノタイピング方法として評価されている。 ここで紹介するプロトコルは、JBGは、野生型cogongrassのものから戻す区別するために、高速かつ信頼性の高いアプローチを提供します。我々は、使用を奨励するこのプロトコルのRSは、このプロトコルの使用に関して得られる結果を報告するために私達に連絡する。このような共有される情報は、JBGのディストリビューションに戻りに関する情報を提供するのに役立ちます。これは、USDAでのレギュレータは非常に侵襲的なcogongrass品種の普及と潜在的なハイブリダイゼーションを回避するために必要されるアクションに、情報に基づいて決定を下すのに役立ちます。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> DNeasy植物ミニキット</td><td>キアゲン、バレンシア、カリフォルニア州</td><td> 69104または69106 </td><td>良い品質のDNAを返す任意の評判の良いゲノム植物DNAキットはこれらの手順については、正常に動作する必要があります。 </td></tr><tr><td> trnF（GAA）-F </td><td>斜めバイオ株式会社</td><td> 3から0578 </td><td>前方に陽性対照用プライマー</td></tr><tr><td> trnL（5 &#39;エクソン）-C </td><td>斜めバイオ株式会社</td><td> 3から0579 </td><td>陽性対照プライマーを逆</td></tr><tr><td> trnLF-C-F1 </td><td>斜めバイオ株式会社</td><td> 3から0864 </td><td>前方に野生型cogongrassプライマー</td></tr><tr><td> trnLF-C-R1 </td><td>斜めバイオ株式会社</td><td> 3から0865 </td><td>逆野生型cogongrassプライマー</td></tr><tr><td> trnLF-R-F2 </td><td>斜めバイオ株式会社</td><td> 3から0866 </td><td>フォワードJBGとプライマーを元に戻すJBG </td></tr><tr><td> trnLF-R-R2 </td><td>斜めバイオ株式会社</td><td> 3から0867 </td><td> JBGとプライマーを元に戻すJBG逆</td></tr><tr><td>利点純PCRのdNTPミックス</td><td> Clontech社、カリフォルニア州マウンテンビュー</td><td> 639125 </td><td></td></tr><tr><td>アドバンテージ2ポリメラーゼ</td><td> Clontech社、カリフォルニア州マウンテンビュー</td><td> 639201または639202 </td><td>優れたプルーフリーディング、ホットスタートTaqポリメラーゼ</td></tr></tbody></table>

a pcr-based genotyping method to distinguish between wild-type and ornamental varieties of imperata cylindrica

野生型I.チガヤ （cogongrass）は負のアフリカ、アジア、ヨーロッパ、ニュージーランド、オセアニア、南北アメリカ1月2日を通して73カ国の農業と天然資源に影響を与え、世界のトップ10最悪の侵略的な植物の一つです。 Cogongrassは、ネイティブの植物種の多種多様な変位と順番に飼料及び避難所のための避難ネイティブ植物種に依存する野生動物を脅かす急速に拡散、monodominantスタンドを形成します。問題は、装飾的な様々な[I.に追加するチガヤ VAR。 チガヤ （レツィウス）]が広くチガヤ &quot;ヤマモモ&quot;、レッドバロン ​​、そして日本人の血草（JBG）の名前で市販されている。この品種は、推定される滅菌と非侵襲的であり、その赤い色の葉を観賞望ましいと考えられている。ただし、適切な条件下では、JBGは、実行可能なシードを生成することができます（キャロルHolko、2009私信）と私は緑に戻すことができます。それは野生型の侵襲的な様々な4（ 図1）の際立った特性を取るように頻繁にcogongrassから区別することはできませんnvasiveフォーム。これはよく訓練された植物分類学者であっても形態に困難なタスクを使用して識別を行います。積極的な緑の表現型にJBGの復帰もまれではありません。コー​​ディングと核と葉緑体DNAの両方の可変領域の配列比較を使用して、我々はJBGは、メリーランド州、サウスカロライナ州とミズーリ州の州内緑の侵襲に戻っていることを確認しています。アクティブcogongrassの侵入がないですJBGは、米国本土ではほぼすべての状態で販売されていると植えられています。よく分かっていないに戻す問題の範囲に戻っ植物は文書化されていないとしばしば破壊されたためです。 この分子プロトコルのアプリケーションが戻り、共起からこれらの品種を保つのを助けることができるJBGを識別する方法を提供するndはおそらくハイブリダイズする。 Cogongrassが嫌気outcrosserであり、異なった遺伝子型と交配する場合、実行可能な生成することができます5月7日広い長距離cogongrassを広げて種子を風を分散。 JBGはcogongrass若干異なる遺伝子型を持っており、cogongrassで実行可能なハイブリッドを形成することができるかもしれません。問題に追加するには、JBGはcogongrass 8月10日よりも寒さと寛容な日陰であり、これら2品種間の遺伝子の流れがより積極的であるハイブリッドを生成する可能性がある、野生型cogongrassより丈夫トレラント、冷陰。野生型cogongrassは、現在、東南アジア、米国で世界中で100万490ヘクタール以上をinfestsながらは50万ヘクタール以上infests、それが急速に北上し、その幅広いニッチや地理的な潜在的な3,7,11のために広がって、米国の大半を占めることができます。遺伝的交差の可能性はUSDA-APHIS連邦有害週間のプログラムのために深刻な懸念である。現在、USDA-APHISは、州WHでJBG禁止しているERE主要なcogongrassの蔓延（例えば、フロリダ州、アラバマ州、ミシシッピ州）があります。 cogongrassとJBG、その分布を拡大しかし、組み合わせから2品種を防止することがより困難に証明することができます。さらに、JBGの配布元に戻すには、現在不明であり、形態を介してこれらの品種を識別する能力なしに、いくつかのcogongrassの蔓延は、JBG戻りの結果であるかもしれません。残念なことに、識別、現在の分子のメソッドは、通常とコストがかかる時間です。どちらもAFLP（増幅断片長多型）と、DNAシークエンシング、依存しています。ここでは、正確にcogongrassとJBG戻す区別する最初の費用対効果と信頼性の高いPCRベースの分子タイピング手法を提案する。

Watch this Scientific Journal Video about A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica at JoVE.com

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

我々は、葉緑体trnL-Fスペーサー領域内の標的DNA配列の違いがの品種を区別することが、様々な特異的PCRプライマーを採用して費用対効果の迅速な分子タイピングプロトコルを提供<em&gt;チガヤ</em&gt;（cogongrass）単独形態によって区別す​​ることができない。これらの品種は、私は、連邦政府が記載されている有害な雑草、cogongrassと密接に関連する、広く普及し、様々な装飾品が含まれてい<em&gt;。チガヤ</em&gt; VAR。<em&gt;チガヤ</em&gt;（日本人の血草）。

の野生型と観賞用品種を区別するPCRベースのジェノタイピング法チガヤ</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

a-pcr-based-genotyping-method-to-distinguish-between-wild-type

Research

JoVE Journal

Biology

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

31.5K ビュー.  The University of Alabama, Huntsville. 我々は、葉緑体trnL-Fスペーサー領域内の標的DNA配列の違いがの品種を区別することが、様々な特異的PCRプライマーを採用して費用対効果の迅速な分子タイピングプロトコルを提供チガヤ（cogongrass）単独形態によって区別す​​ることができない。これらの品種は、私は、連邦政府が記載されている有害な雑草、cogongrassと密接に関連する、広く...

ビデオ: A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

生物学

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

タンパク質のメンブレンオーバーレイアッセイ：可溶性および不溶性タンパク質間の相互作用をテストするためのプロトコル in vitroで</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

マイコプラズマの最適化されたPCRベースの検出

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

アイスキャップ：成長させる方法シロイヌナズナ</emハイスループットジェノタイピング用96ウェルプレートで>とトマト

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

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 ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

大規模なSNPジェノタイピングアプリケーション用インフィニウムアッセイ

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高スループット、Sc2.0ジェノタイピングのためのリアルタイムPCRアッセイ

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

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 ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

電気穿孔法によるマウス膀胱尿路上皮にプラスミド DNA 配信法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

の野生型と観賞用品種を区別するPCRベースのジェノタイピング法チガヤ

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この動画の章

パイロシーケンシング：正確なジェノタイピングのための簡単な方法

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

タンパク質のメンブレンオーバーレイアッセイ：可溶性および不溶性タンパク質間の相互作用をテストするためのプロトコル in vitroで

マイコプラズマの最適化されたPCRベースの検出

アイスキャップ：成長させる方法シロイヌナズナとトマト

前のDNA精製せずに植物や動物のサンプルの遺伝子型

大規模なSNPジェノタイピングアプリケーション用インフィニウムアッセイ

qPCRTag分析 - 高スループット、Sc2.0ジェノタイピングのためのリアルタイムPCRアッセイ

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

電気穿孔法によるマウス膀胱尿路上皮にプラスミド DNA 配信法

の野生型と観賞用品種を区別するPCRベースのジェノタイピング法チガヤ

要約

さらに動画を探す

この動画の章

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

タンパク質のメンブレンオーバーレイアッセイ：可溶性および不溶性タンパク質間の相互作用をテストするためのプロトコル in vitroで

マイコプラズマの最適化されたPCRベースの検出

アイスキャップ：成長させる方法シロイヌナズナとトマト

前のDNA精製せずに植物や動物のサンプルの遺伝子型

大規模なSNPジェノタイピングアプリケーション用インフィニウムアッセイ

qPCRTag分析 - 高スループット、Sc2.0ジェノタイピングのためのリアルタイムPCRアッセイ

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

電気穿孔法によるマウス膀胱尿路上皮にプラスミド DNA 配信法

パイロシーケンシング：正確なジェノタイピングのための簡単な方法