Name: small-scale extraction of caenorhabditis elegans genomic dna
Uploaded: 2022-06-07T13:00:00
Description: Watch this Scientific Journal Video about Small-Scale Extraction of Caenorhabditis elegans Genomic DNA at JoVE.com

N2菌株和 大肠杆菌 OP50细菌是从 Caenorhabditis 遗传学中心（CGC）获得的，该中心由NIH研究基础设施计划办公室（P40 OD010440）资助。 blmp-1（tm548） 菌株来自日本国家生物资源项目。作者感谢WormBase。这项工作得到了NIH R01GM097591对T.L.G.的支持，德克萨斯女子大学对T.L.G.的内部资助，以及TWU的学生研究中心对M.F.L.的支持。

1. 秀丽隐杆线虫的 维持
注意：N2（野生型）和 blmp-1（tm548）C.秀丽隐杆线虫 菌株在20°C下维持在标准线虫生长培养基（NGM）板上。
<ol><li>通过将23.005gNGM粉末溶解在2L烧瓶中的973mL水中来制备NGM板。用铝箔（或允许排气的可高温高压灭菌盖）盖住烧瓶开口，并用高压灭菌胶带固定盖子。高压灭菌器溶解粉末并以至少30分钟的灭菌循环对内容物进行灭菌?...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+SMA-10/LRIG+is+a+conserved+transmembrane+protein+that+enhances+bone+morphogenetic+protein+signaling.">Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling.</a> PLoS Genetics. 6, 1000963 (2010).</li><li>Nelson, M. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLMP-1/Blimp-1+regulates+the+spatiotemporal+cell+migration+pattern+in+C.+elegans.">BLMP-1/Blimp-1 regulates the spatiotemporal cell migration pattern in C. elegans.</a> PLoS Genetics. 10, 1004428 (2014).</li><li>Lakdawala, M. F., Madhu, B., Faure, L., Vora, M., Padgett, R. W., Gumienny, T. 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确定 秀丽隐杆线虫 的基因型是进行遗传杂交以创建新的 秀丽隐杆线虫 菌株的重要步骤。使用单个或几个 秀丽隐杆线虫进行 基因组DNA提取是秀 丽隐杆线虫基因分型的关键步骤。该协议描述了使用商业试剂盒从 秀丽隐杆线虫 中提取基因组DNA。此方法速度快，工作可靠。使用这种方法提取的基因组DNA可用于下游应用，包括基因分型，测序和克隆。所描述的DNA提取?...

在这里，提出了两种用于秀 丽隐杆线虫 裂解的相关方案，以使DNA可用于基于PCR的应用。PCR是一种常用的分子技术，用于许多应用，包括用于克隆和测序的DNA片段的基因分型和扩增等。小（1毫米），自由生活的蛔虫 秀丽隐杆线虫 是生物学研究的流行动物系统。从单个动物或少数动物中获得合适的基因组DNA足以通过PCR扩增序列。晚期L4幼虫和成虫在 子宫内仅含有约1，000个体细胞（包括一些多核多倍体细胞），生殖细胞和（如果动物是妊娠雌雄同体）后代。然而，这些动物受到角质层的保护，必须破坏角质层以提取基因组DNA2。制备用于PCR的线虫基因组DNA模板的标准方法涉及多个步骤，需要几个小时。首先将动物在含有蛋白酶K（−70°C或更低）的蠕虫裂解缓冲液中冷冻至少15-45分钟（某些方案建议更长）3，4，5，6。这一步打开了动物。
冷冻后，将动物在60-65°C下孵育1小时以使蛋白酶K起作用，然后将酶在95°C下灭活15-30分钟。 蛋白酶K破坏降解DNA的核酸酶。在PCR之前蛋白酶K失活对于防止蛋白酶K降解DNA聚合酶非常重要。这里描述的两种基于试剂盒的方案是从单个动物或几个线虫中提取基因组DNA的快速，可靠且具有成本效益的方法，用于日常研究和教学实验室应用。所使用的试剂盒最初由制造商优化，以从动物组织，唾液和毛发中提取DNA7。它使用专有的组织制备溶液和提取溶液来裂解细胞并使基因组DNA易于访问。然后，专有的中和溶液中和可能抑制PCR的组分（例如，盐，离子和Mg2 +结合分子）。
在基因分型时，可以测试单个动物。在确定菌株是否纯合时，从单个动物中测试六个或更多后代可以高度肯定一个品系是否纯合（从杂合亲本随机挑选六个纯合突变后代的几率为0.02%[（1/4）6 ×100%= 0.02%]）。该方法1）简单明了，步骤比蛋白酶K方法少，2）将模板制备时间减少到15分钟。这项工作的结果表明，所开发的方案在从单个或几个蠕虫中提取基因组DNA方面具有强大的作用，这些基因组DNA可以可靠地用于不需要高度纯化的DNA的下游应用，包括PCR。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>autoclave tape</td>
			<td>Defend</td>
			<td>43237-2</td>
			<td></td>
		</tr>
		<tr>
			<td>aluminium foil, heavy duty</td>
			<td>Reynolds Wrap</td>
			<td>2182934</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride</td>
			<td>Millipore Sigma</td>
			<td>102382 (CAS 10035-04-8)</td>
			<td></td>
		</tr>
		<tr>
			<td>Extract-N-Amp kit (includes Tissue Preparation Solution, Extraction Solution, and Neutralization Solution)</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>XNAT2-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp hotplate/stirrer</td>
			<td>Fisher Scientific</td>
			<td>11-100-495H</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media, Lennox, capsules</td>
			<td>MP Biomedicals, LLC</td>
			<td>3002-131</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate, 97% pure, anhydrous</td>
			<td>Thermo Scientific</td>
			<td>AC413485000 (CAS 7487-88-9)</td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge</td>
			<td>Labnet International, Inc.</td>
			<td>PrismR, C2500-R</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB Q5U Hot Start High-Fidelity DNA polymerase</td>
			<td>New England Biolabs, Inc.</td>
			<td>M0515S</td>
			<td>&#34;Pol E&#34; used in Supplemental Figure S1, a high-speed, high-fidelity polymerase (20&#8211;30 s/kb)</td>
		</tr>
		<tr>
			<td>NGM media powder</td>
			<td>US Biological Life Sciences</td>
			<td>N1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>New England Biolabs, Inc.</td>
			<td>M0531S</td>
			<td>&#34;Pol D&#34; in Figure 1B, a high-speed, high-fidelity polymerase (15&#8211;30 s/kb)</td>
		</tr>
		<tr>
			<td>primers</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeSTAR GXL polymerase</td>
			<td>Takara Bio Inc.</td>
			<td>R050B</td>
			<td>&#34;Pol C&#34; in Figure 1B, a high-fidelity polymerase (1 min/kb) for GC-rich templates and templates up to 30 kb</td>
		</tr>
		<tr>
			<td>Quick-Load Purple 2-log DNA Ladder (0.1&#8211;10.0 kb)</td>
			<td>New England Biolabs, Inc.</td>
			<td>N0550S</td>
			<td></td>
		</tr>
		<tr>
			<td>SapphireAmp Fast PCR Master Mix</td>
			<td>Takara Bio Inc.</td>
			<td>RR350A</td>
			<td>&#34;Pol A&#34; in Figure 1B, polymerase used in Figure 1A, C, D, a high-speed polymerase (10 s/kb) for targets up to 5 kb</td>
		</tr>
		<tr>
			<td>Sigma ReadyMix Taq PCR reaction mix</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>P4600</td>
			<td>&#34;Pol B&#34; in Figure 1B, a polymerase (1 min/kb) for targets up to 7 kb</td>
		</tr>
		<tr>
			<td>SimpliAmp thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>A24812</td>
			<td></td>
		</tr>
		<tr>
			<td>stir bar</td>
			<td>Fisher Scientific</td>
			<td>14-512-126</td>
			<td></td>
		</tr>
		<tr>
			<td>vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>worm pick</td>
			<td>Genesee Scientific Corporation</td>
			<td>59-AWP</td>
			<td></td>
		</tr>
	</tbody>
</table>

small-scale extraction of caenorhabditis elegans genomic dna

从单个或几个 秀丽隐杆线虫 中提取基因组DNA具有许多下游应用，包括用于基因分型系，克隆和测序的PCR。传统的基于蛋白酶K的方法从 秀丽隐杆线虫 中提取基因组DNA需要几个小时。有效打开 秀丽隐杆线虫 角质层和提取基因组DNA的商业提取试剂盒是有限的。本文介绍了一种简单，快速（约15分钟）且具有成本效益的提取 秀丽隐杆线虫 基因组DNA的方法，该方法适用于课堂和研究应用。这种DNA提取方法经过优化，使用单个或几个晚幼虫（L4）或成体线虫作为获得可靠模板进行PCR的起始材料。结果表明，DNA质量适用于通过PCR扩增不同大小的基因靶标，即使在每次反应中稀释到单个成虫DNA的五十分之一时，也可以对单个或少数动物进行基因分型。所报告的实验方案可以可靠地用于从单个或一小个 秀丽隐杆线虫 样本中快速生成DNA模板，用于基于PCR的应用。

使用商业试剂盒或传统裂解方案从单个或几个野生型成虫中提取基因组DNA，以比较这两种方法的疗效。然后将这些裂解物用作PCR的模板，以扩增〜2，100 bp的较大靶标（编码 blmp-1）或〜500 bp的较小靶标（编码 sma-10的一部分）。两种方法都成功产生了适当的PCR产物（图1A）。
接下来，证明了试剂盒提取的基因组DNA作为各种DNA聚合酶模板的能力...

Watch this Scientific Journal Video about Small-Scale Extraction of Caenorhabditis elegans Genomic DNA at JoVE.com

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

这里介绍的是使用市售组织试剂盒从一只或几只动物中直接和相对快速地分离 秀丽隐杆线虫 基因组DNA的描述。所得的gDNA制剂是PCR的合适模板。

小规模提取 秀丽隐杆线虫 基因组DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...

我们开发了一种简单，快速且具有成本效益的方法来提取秀丽隐杆线虫基因组DNA，非常适合课堂和研究应用。这些实验方案可以可靠地用于从单个或一小部分C.elegance样品中快速生成DNA模板，用于基于PCR的应用。没有选择蠕虫或使用微管道的经验的用户可能会在需要这些技能的步骤中遇到困难。观看熟练的从业者和练习可能会有所帮助。演示该程序的将是来自我实验室的高级研究生Farhan Lakdawala。为了从单个秀丽隐杆线虫中提取DNA，首先，设置裂解程序，包括一个循环在55摄氏度下10分钟，然后在95摄氏度下在热循环仪中三分钟。为了制备裂解液，首先，从试剂盒中加入0.8微升的提取溶液到冰上0.2毫升PCR管的内壁。然后从试剂盒中加入0.2微升的组织制备溶液到提取溶液的液滴中，并通过peting混合。通过选择蠕虫进行DNA提取，首先，在解剖显微镜下鉴定L4或成年动物。L4雌雄同体可以通过特征性外阴来识别，该外阴在动物中间可见为苍白的半圆。要识别成年雌雄同体，请选择平板上最大的动物之一，该动物的子宫中可能有可见的椭圆形胚胎。接下来，使用铂丝镐，将选定的动物转移到溶液中。要收集管底部的内容物，请在室温下以2000×g离心管两到三秒钟。然后将试管放入热循环仪中，并运行先前设置的裂解程序。此裂解程序结束后，短暂离心管并将其放在冰上。然后从试剂盒中加入0.8微升中和溶液，并通过移液混合。再次，在室温下以2000×g离心管两到三秒钟。如果不立即使用裂解液，请将管子储存在四摄氏度以备将来使用。为了从单个蠕虫中提取DNA，首先，将一个周期的裂解程序设置为55摄氏度10分钟，然后在热循环仪中95摄氏度下进行三分钟。接下来制备裂解液，方法是首先将两微升的提取溶液从试剂盒中加入到冰上0.2毫升PCR管的内壁上，然后从试剂盒中加入0.5微升的组织制备溶液到提取液滴中，然后通过移液混合内容物。识别L4或成年动物后，使用铂丝镐将足够数量的动物转移到溶液中。接下来，在室温下以2000×g离心管两到三秒钟，然后将管放入热循环器中并运行先前设置的裂解程序。程序完成后，短暂离心管并将其放在冰上。然后将两微升中和溶液从试剂盒中加入到管中并通过移液混合。再次，在室温下以2000×g离心管两到三秒钟。如果不立即使用，请将裂解液储存在四摄氏度以备将来使用。通过方案提取的基因组DNA可以成功地用作PCR模板，商业试剂盒和传统的蛋白酶K方法都产生了与2100碱基对产品和500碱基对产物具有相似PCR扩增水平的DNA。用于DNA裂解的试剂盒方法还提供了对多种DNA聚合酶有效的PCR模板。即使在不同的稀释度下，商业试剂盒从单个动物中提取的DNA也支持以同等效率扩增2100碱基对DNA片段的PCR扩增。然而，500个碱基对DNA片段的扩增随模板浓度而变化。从单个动物或从多个动物中提取的DNA作为测定基因型的PCR模板的适用性通过基因及其突变变异的成功扩增进一步证明了。由于该方案涉及移液少量试剂，为确保一致性，请使用预混液进行多次反应，良好的管道技术和校准良好的移液器。此外，旋转管中的内容物可确保适当的裂解。这种方法可以放大，适用于提取秀丽隐杆线虫基因组DNA用于其他下游应用，并可用于从其他线虫物种中提取DNA。鉴于该协议的简单性，速度，鲁棒性和低成本，它非常适合时间和资源有限的研究和课堂应用。

Research

JoVE Journal

Biology

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

分离和<em在体外激活线虫精子

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

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

小规模提取 秀丽隐杆线虫 基因组DNA

小规模提取秀丽隐杆线虫基因组DNA