排せつのマッサージと早期支援ありがとうサンノゼ施設と研究所のジェフリー ・ Kittilson をサポートします。この作品は、BJH に ND EPSCoR FAR0022429 賞によって支えられました。出版費用は寛大清心生物学科によってをサポートされていました。

このプロトコルは脊椎動物の動物科目を含み、機関動物ケアおよび使用委員会 (IACUC) ノースダコタ州立大学でによって承認されています。
1. 精液採取排せつマッサージを使用してスズメ目の鳥から
注: 排せつマッサージ繁殖アクティブな鳥のみ、効果的な精液収集手法ですが、適切な飼育状況で繁殖シーズン以外正常に実行することができます。排せつのマッサージを使用する前にターゲット種の生殖活動を決定してください。キャプチャ時に野生の鳥や鳥が飼育下で開催、この手法を使用することができます。
<ol><li>(繁殖条件) で、雄の鳥から始まる非利き手でのバンダのグリップは、腹側 (図 1) の手のひらに触れると支配的な手に鳥を転送します。<ol>
<li>鳥の頭は手のひらの外側の縁に最も近いことを確認します。軽く頭と小指、リングおよび中指を使用してボディを保護します。これは、鳥の口と尾公開を残します。</li>
		<li>緩んでいるか、軽く手のひらを使用して拘束された鳥の足を残します。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> 図 1: 排せつマッサージ グリップします。鳥はその腹側の手のひらと小指担保の頭に触れると、軽く利き手でセキュリティ保護されます。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol start="2"><li>腔の優れた終わりのどちら側でも支配的な人差し指と親指を置き、軽く尾 (図 2) の方の非常にわずかな圧力を適用するときに一緒に指を挟んだり。排泄腔が片脚、排せつの粘膜 (ピンク インテリア) を公開します。 注: 鳥がない場合排せつの突起が小さいことができる現在、精液を生産と排泄腔が片脚にくい。経験を持つ、繁殖アクティブおよび非アクティブの男性5,11cloacas 区別するために簡単です。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> 図 2: 位置や排せつ突起を指します。優れたエンド (A) にわずかな圧力を適用することによって、総排出腔を片脚します。繁殖アクティブな男性のスズメ目、排せつの突起は明白と (B) の会社。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol start="3"><li>非支配的な手で手を安定させるために尾のベースの背側に指のパッドを配置し、すぼまり腔下親指の先端。 注: 短く、滑らかなサムネイルは排せつマッサージを実行するため有利であり傷害から鳥を保護します。</li>
	<li>中型圧力、クロアカ以下の滞在の反復運動に深いと頭側非利き手親指を移動します。鳥が射精するまで続行します。個人によって通常 1 といくつかのダース ストローク間かかります。</li>
	<li>鳥が射精すると、すぐに microhematocrit チューブで精液を収集します。複数射精を取得する排せつマッサージを繰り返します。 注: スズメ目精液は明るい茶色で、通常やや濃厚。白や暗い物質排泄腔から排出、精液は、精液のサンプルを汚染する可能性があります。腔に少量の血液の外観または混合される精液は、通常のマッサージ中にあまりにも多くの圧力によって引き起こされます。</li>
	<li>DNA の抽出の 1x PBS (pH 7.4) の 20 μ L の微量遠心チューブに精液を配置します。注: 1 × PBS で保存精液人工授精には適していませんが、後日 DNA 抽出のため使用することができます-80 ° C で保存する場合PBS 室温または冷蔵しますが、精液のサンプルが到着したとき、液体の状態にする必要があります。<ol>
<li>サンプルで精子の存在を確認するには、複合顕微鏡 (図 3) の下で希釈サンプルの少量を表示します。</li>
	</ol>
</li>
	<li>抽出まで-80 ° C で希釈精液を格納します。</li>
</ol>2. 鳥の精液からの DNA の抽出
注: このプロトコルは、複数の商業 DNA 抽出キットでテストされていますが、のみ 1 つの DNA キット12で使用する場合は成功しています。キットのプロトコルからの変更手順で示されます *。
<ol><li>ジチオトレイトール (DTT) 水での 1 M 溶液を準備し、vortex.* を使用してミックス<ol>
<li>因数 DTT ソリューション使用直前まで-20 ° C で保存します。抽出する各サンプルは 1 M の DTT の solution.* の 10 μ L を必要とします。</li>
	</ol>
</li>
	<li>20 ミリメートル pH 8.0; トリス-の Cl を含むバッファーを準備します。20 mM EDTA;200 mM の NaCL;4 %sds。各サンプルを抽出するこの buffer.* の 90 μ L が必要です。<ol>
<li>室温でバッファーを保存します。それは沈殿させた場合 dissolve.* 56 ° C に温めてください。</li>
	</ol>
</li>
	<li>ロッカーまたは 65 にシェーカーを含むインキュベーターを予熱 ° C.*</li>
	<li>すぐに抽出を開始、する前にバッファーの場合は 10 試料を抽出手順 2.2 各サンプル抽出する、例えば900 μ L バッファーに加え 100 μ L DTT ソリューションで準備の 90 μ L で 10 μ L DTT ソリューションをミックスします。この混合物は DTT buffer.* と呼ばれる、</li>
	<li>全体の希釈精液サンプルを 70 μ L 1 × PBS を追加し、vortex.* でよく混ぜ</li>
	<li>100 μ L を追加 DTT バッファー、サンプルに 10 μ L プロティナーゼ K ソリューション。20 秒間、渦、予熱したインキュベーターで 1 時間インキュベート ロッカーまたは shaker.*</li>
	<li>200 μ L バッファー アルと 200 μ L を追加新鮮なエタノール (100%)、20 seconds.* の渦</li>
	<li>スピン列にサンプル全体を転送し、6,000 x g. 破棄で 1 分コレクション チューブを遠心分離します。</li>
	<li>新しいチューブに列を配置し、500 μ L 準備バッファー AW1 を追加します。6,000 x g. 破棄で 1 分コレクション チューブを遠心分離機します。</li>
	<li>新しいチューブに列を配置し、500 μ L 準備バッファー AW2 を追加します。6,000 x g に 1 分間遠心は、フロースルーと列がチューブに戻って配置を破棄します。</li>
	<li>20,000 x g 破棄に 3 分コレクション チューブを遠心し、1.5 mL 遠心チューブに列を配置します。</li>
	<li>ピペット 35 μ L AE の列膜に直接バッファーし、5 分ではあ、室温でインキュベート</li>
	<li>スピン列 14,000 x g 破棄に 1 分を遠心し、-80 ° C で抽出されたサンプルを格納</li>
</ol>

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cornell Lab of Ornithology Handbook of Bird Biology, 3rd Edition. , (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QIAamp DNA Micro Handbook. , (2014).</li><li>Carter, K. L., Robertson, B. C., Kempenaers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differential+DNA+extraction+method+for+sperm+on+the+perivitelline+membrane+of+avian+eggs.">A differential DNA extraction method for sperm on the perivitelline membrane of avian eggs.</a> Mol. Ecol. 9, 2149-2154 (2000).</li><li>Birkhead, T. R., Buchanan, K. L., Devoogd, T. J., Pellatt, E. J., Catchpole, C. 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著者は、彼らは競合する金銭的な利益があることを宣言します。

シンプルで信頼性の高い繁殖アクティブ状態でない中小鳥から精液を収集し、鳥の精子から DNA を抽出法について述べる.
記述されていた精液の DNA の抽出のプロトコルは便宜上、キットからは変更が、鳥ザーメン使用の洗練されました。精子が標準抽出化学9,13、溶解耐性と産スズメ目鳥から収集した精液のサンプルが非常に小...

鳥が生物学の研究に幅広く使用されている精子は排せつを使用して簡単にサンプリングすることができますと精子の質と競争1、精子テロメア ダイナミクス2エピジェネティクス3と似たようなトピックを含む研究のための理想的な科目マッサージ。排せつマッサージは鳥4,5,6から精液を収集するための穏やかな、非侵襲的な手動技術です。繰り返しサンプルを簡単に得ることができる、特別な工具も不要、簡単にフィールドまたはラボで実行できるように。排せつのマッサージは、何十年も使用されている、利用可能な説明が書かれてから学ぶことは困難です。このパブリケーションは、排せつマッサージを学習に関わる不確実性を低減するものです。
排せつマッサージを使用して鳥から収集した精液は、運動評価4,7または人工授精8、すぐに使用または高度なイメージングおよび DNA の抽出等の用途として冷凍することができます。スズメ目の鳥から精液サンプルは小さいが密集精子を含まれています。精子9の特殊な保護機能を克服するための変更を簡単にするため商業抽出キットを使用して DNA を抽出します。抽出後、qPCR10を使用して精子テロメアを測定できます。

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microhematocrit capillary tubes</td>
			<td style="width:71px;">Fisherbrand</td>
			<td style="width:119px;">22-362566</td>
			<td style="width:167px;">Any supplier can be used. Smaller tubes may be more effective for collection of small semen samples, such as from very small passerine birds like chipping sparrow.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tubes</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1x PBS</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td>Used for storing semen samples prior to DNA extraction.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris base</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">10708976001</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1233508</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">793566</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SDS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1614363</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA micro kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">56304</td>
			<td>Qiagen QIAamp DNA micro kit is recommended specifically. This DNA extraction protocol has been attempted with other commercial kits but with little success. The reasons for this are unknown to us.&#160;</td>
		</tr>
	</tbody>
</table>

avian semen collection by cloacal massage and isolation of dna from sperm

精液の採取は、精子の質、精子テロメア ダイナミクス、エピジェネティクス研究を含むアプリケーションの広い範囲のために役立ちます。鳥は広く使用されている科目は、生物学的研究、繰り返し精子サンプルを含む研究に最適です。ただし、いくつかのリソースは現在利用可能な収集および鳥の精子から DNA を抽出する方法を学びたい人のためです。ここで排せつマッサージ、鳥精子を収集するための穏やかな、非侵襲的な手動技術について述べる。文献でこの手法を確立すると、使用可能な記述から学ぶことは困難ことはできます。また、変更と商業抽出キットを使用して鳥の精液から DNA を抽出するための情報を提供いたします。排せつマッサージは、生殖状態で中規模男性鳥で容易に使用することができます。次のコレクション、精液は運動アッセイのためすぐに使用または記載プロトコルを次の DNA の抽出のために冷凍できます。この抽出プロトコル鳥精子の洗練された、いくつかのスズメ目種 (通行人国立公園、 Spizella passerina、 Haemorhous メキシコ、およびつぐみ migratorius から収集されたサンプルで正常に使用されています。) と 1 つの columbid (コルンバ ・ リヴィア)。

排せつマッサージと DNA 抽出を使用していくつかのスズメ目種と 1 つの columbid (表 1) で行われた記述のプロトコル。排せつマッサージによって収集された精液に精子の存在は、400 倍の倍率 (図 3) で少量の化合物顕微鏡下で希釈した試料を表示することによって確認されました。ここで説明されているプロトコルを使用して家すずめ?...

Watch this Scientific Journal Video about Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm at JoVE.com

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

このプロトコルでは、排せつマッサージ、鳥、および変更と商業抽出キットを使用して鳥の精子から DNA の抽出から精液を収集するため非侵襲的な手動技術について説明します。

排せつマッサージと精子から DNA の隔離による鳥の精液採取

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

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JoVE Journal

Biology

15.8K ビュー.  North Dakota State University. このプロトコルでは、排せつマッサージ、鳥、および変更と商業抽出キットを使用して鳥の精子から DNA の抽出から精液を収集するため非侵襲的な手動技術について説明します。

ビデオ: Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

収穫精子とマウスの人工授精

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

生物学

Isolation of Genomic DNA from Mouse Tails

マウスの尾からのゲノムDNAの単離

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

人間の膵島単離：パートI：膵組織の消化とコレクション

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

鳥類胚弁膜前駆細胞の単離と培養

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

アイソレーションと試験管内で</emの>活性化線虫（Caenorhabditis elegans）精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

血漿タンパク保存用採血キットからゲノムDNAを抽出するために実用的で新手法

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

当然のことながら環境から磁性細菌の発生の収集、分離、濃縮

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

マウスモデルにおける子宮卵管胚移植と精管切除

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

培養<em&gt;線虫（Caenorhabditis elegans）</em &gt;微小粒子衝撃による無菌液体培地およびトランスジェニックワームの作成に

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNAエレクトロポレーション、筋線維の単離およびイメージング

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

排せつマッサージと精子から DNA の隔離による鳥の精液採取

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