在我们的实验室的工作是事业奖由美国国家科学基金会和NIGMS，美国国立卫生研究院授予R15GM86839区MCB0551798支持。

1。增长和紫外线照射。 <ol><li> 200μL新鲜隔夜生长在戴维斯中型1辅以葡萄糖0.4％，0.2％casamino酸，和10微克/毫升胸腺嘧啶（DGCthy中型）和100μg/ mL氨苄青霉素质粒pBR322的文化沉淀。然后，细胞沉淀悬浮于200μLDGCthy缺乏氨苄青霉素介质和使用的预防针DGCthy中等20毫升。 </li><li>文化是生长没有氨苄青霉素选择一个摇床中于37 ° C为0.5外径600（〜5 × 10 8细胞/毫升）。没有氨苄青霉素增长避免了对异常或非生产性的复制中间体中可能出现的一些突变体的选择。此外，如果用紫外线光诱导损伤，从媒体清除氨苄青霉素是必要的，因为它吸收了强烈的细胞在这些波长和盾牌，减少紫外线的有效剂量。 </li><li>黄色的灯光下工作，文化是一个躁动的旋转平台上放置在直径15厘米的培养皿。我们的文化是放置在一个15瓦杀菌灯产生的〜1 J/m2/sec，这是使用紫外线光度计测量的照射量率的距离。文化是照射50 J/m2然后立即放回晃动在实验期间的37℃培养箱中培养。此剂量产生的，平均1环丁烷嘧啶二聚体每4.5 kb的单链DNA。黄色灯光防止光解环丁烷嘧啶二聚体的光复活逆转。 </li></ol> 2。 DNA的提取。 <ol><li>在复制中间体进行审查的时候，文化0.75毫升等分放入0.75毫升冰冷NET30缓冲区（pH值8.0，100毫米氯化钠，10毫米三，30毫米EDTA），并置于冰上，直到结束日久。我们通常运行90分钟的时间当然，研究样本为0，15，30，45，60和90分钟。 EDTA和低温的服务，以有效地制止复制和核苷酸切除修复。 </li><li>然后每个样品颗粒，悬浮于150μL1.5 mg / ml的溶菌酶和0.2 mg / ml的TE RNaseA（10毫米三，pH值8.0，1 mM EDTA的），并溶解在37 ° C为20分钟。此时，加入10μl蛋白酶K（10mg/ml的），并加入10μl20％sarkosyl的添加和孵化允许持续1小时。蛋白酶K和sarkosyl有助于释放酚抽提发生之前，可能与膜蛋白相关的DNA片段。由于积极DNA复制蛋白或膜复合物往往是必然的，这有助于增加产量恢复的复制片段。 </li><li>每个样品中加入2卷苯酚和管轻轻倒5分钟，然后提取样品。然后氯仿/异戊醇（24 / 1）2卷管轻轻再次倒立5分钟。 </li><li>在14000 RPM离心5分钟，每个样品的水相在离心样品被删除，并放置到一个新的管。然后添加4卷氯仿/异戊醇（24 / 1），管轻轻倒5分钟，再离心5分钟14,000转。 </li><li>顶部，每个样本中的水相，然后透析1小时，发现一个47毫米的Whatman 0.025微米孔径的硬盘，250毫升的0.1X TE漂浮在一个烧杯，每个样品100μL。由于更容易受到剪切，复制与单链地区或分支点的结构是我们通常切割用刀片枪头口更宽，并尽量减少剪切力。在一般情况下，移液应保持到最低限度，直到后的DNA用限制性内切酶消化。 </li><li>每个样本，然后消化PvuII（New England Biolabs公司）线性化质粒的复制起点的下游。 </li><li>在琼脂糖凝胶，100μL氯仿和6X样染料含有溴酚蓝和二甲苯蓝20μL加载之前被添加到每个样品和混合。然后，水样染料相40μl装入凝胶。为了最大限度地减少结构性偏差，结构构件和DNA剪切，这个分离过程中不包括任何的步骤，以丰富单链地区，沉淀，或集中的DNA样本，下面的细胞裂解。 </li></ol> 3。双向凝胶电泳和Southern杂交分析。 <ol><li> 2维的琼脂糖凝胶电泳分析，修改3。第一维，限制的DNA样本是运行在1V/cm 1X TBE通过0.4％琼脂糖凝胶。一公升的10X TBE原液包含： <ul><li> 108克三基地</li><li> 55克硼酸</li><li> 40毫升0.5M EDTA（pH值8.0） </li></ul></li><li>一个lambda的HindⅢ大小标记是在第一线加载，然后装入样品在每一个其他车道。我们通常运行的第一ðimension〜12-15小时。跳绳车道，使得更容易切片车道投中第二个维度。低电压和低％琼脂糖凝胶电泳分离的DNA片段的大小主要取决于服务。 </li><li>对于第二个层面，切胶道第一维凝胶使用一个大的屠刀。第一线包含的lambda的HindⅢ标记可以用溴化乙锭染色。为指导，作物使用的lambda的HindⅢ标记和丢弃凝胶低于线性质粒，预计运行的地区。在这些条件下，我们发现，线性pBR322的运行略高于二甲苯蓝染色。 </li><li>投第二个维度，每个泳道是横放跨越了一个空的凝胶施法的顶部。准备一个解决方案的1.0％琼脂糖1X TBE并冷却至55 ° C。此时，凝胶溶液倒在投第二个层面，以确保完全覆盖凝胶片。一旦凝胶，它是在6.5V/cm运行在电泳单位，允许循环缓冲区。我们通常运行的第二个维度〜5.5-7小时。有效的高电压和高％琼脂糖凝胶电泳分离的DNA片段根据其形状，以及大小。通过第二个维度，非线性形状运行更慢。 </li><li>以下电泳，凝胶，然后在H 2 O的冲洗，洗两次在0.25米盐酸400毫升15分钟，与H 2 O冲洗，然后两次在400毫升0.4M的NaOH冲洗30分钟。酸清洗服务成较小的片段，更有效地转移部分缺口的DNA分子。这一步是必要的，因为大尺寸和不同寻常的形状DNA复制中间体。 </li><li>凝胶的DNA，然后转移到一个Hybond ñ +尼龙膜 4 。我们用0.4M氢氧化钠碱向下传输系统。简单地说，两片在0.4M NaOH溶液中浸泡吸墨纸放在大栈的纸巾。尼龙膜浸泡在0.4M氢氧化钠和放在吸墨纸上。在此之上，然后仔细分层凝胶的NaOH溶液中浸湿，另一块吸墨纸。最后，一​​个长片湿面纸是分层的顶部和其两端放置一个包含1 L 0.4M NaOH溶液作为灯芯的菜肴。我们通常让DNA转移为6-12小时。 </li><li>尼龙膜被删除，在5X SSC缓冲液洗20-30秒，并在10.5毫升预杂交溶液杂交辊瓶放置。然后，膜孵育在42℃，旋转超过6小时。预杂交的解决方案： <ul><li> 5.0毫升甲酰胺</li><li> 0.5毫升，20％SDS </li><li> 2.5毫升20X SSC * </li><li> 2.0毫升50X登哈特* </li><li> 0.5毫升鲑鱼精DNA（10mg/ml的） * SSC和登哈特的食谱中可以找到5 </li></ul></li><li>在预杂交期间，1微克质粒质粒缺口平移标记由罗氏制药公司使用标有[32 P] - dCTPα-提供的协议与32P。放射性标记的探针（100μL）是变性孵化在98 ° C为10分钟一个螺丝帽的离心管中，然后立即放在冰上。 </li><li>变性的探针加入5.9毫升的杂交液，其中包含： <ul><li> 3.0毫升甲酰胺</li><li> 1.5毫升南南合作</li><li> 1.0毫升H 2 O </li><li> 0.15毫升50X登哈特的</li><li> 0.10毫升20％的SDS </li><li> 0.15毫升10mg/ml的鲑鱼精DNA </li></ul></li><li>预杂交的解决方案是泼出去的滚瓶，滚瓶，然后返回到42℃的孵化器和旋转超过12小时以上，杂交液浇入。 </li><li>杂交液是泼出去的滚瓶。然后，印迹洗20分钟〜150毫升一洗0.5X SSC，0.1％SDS在42 ° C的解决方案与旋转的辊瓶的4倍。 </li><li>膜是最后一次洗涤后，放在纸巾上，直到液体已明显消失。然后将滤膜聚氯乙烯保鲜膜包裹在接触到phosphorimager屏幕。我们可视化和量化的放射性，使用风暴840及其相关ImageQuant软件。 </li></ol> 4。代表性的成果： <img src="/files/ftp_upload/2220/2220fig1.jpg" alt="图1" /> 图1。质粒复制中间体的存在和由紫外线引起的DNA损伤的情况下观察。PvuII迁移模式消化质粒质粒2D -琼脂糖凝胶电泳分析观察图解。非复制的线性质粒作为一个线性的4.4 kb片段运行。复制的质粒，比非复制的片段，由于其尺寸较大的迁移速度较慢并没有形成Y形结构nlinear形状。这种迁移模式，形成一道弧线，从线性区域扩展以及对。紫外线照射后，双Y或X形分子观察锥地区的迁移速度比Y形分子的弧线。用32P标记的pBR322的探测的二维凝胶南分析的一个例子是细胞紫外线照射和紫外线照射（从出版商的许可复制）后15分钟后立即显示。

<ol>
	<li>Davis, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Isolation+of+Biochemically+Deficient+Mutants+of+Bacteria+by+Means+of+Penicillin.">The Isolation of Biochemically Deficient Mutants of Bacteria by Means of Penicillin.</a> Proc Natl Acad Sci U S A. 35, 1-10 (1949).</li><li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Damage-Induced+Replication+Fork+Regression+and+Processing+in+Escherichia+coli.">DNA Damage-Induced Replication Fork Regression and Processing in Escherichia coli.</a> Science. 299, 1064-1067 (2003).</li><li>Friedman, K. L., Brewer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+replication+intermediates+by+two-dimensional+agarose+gel+electrophoresis.">Analysis of replication intermediates by two-dimensional agarose gel electrophoresis.</a> Methods Enzymol. 262, 613-627 (1995).</li><li>Spivak, G., Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+damage+and+repair+in+specific+DNA+sequences.">Determination of damage and repair in specific DNA sequences.</a> METHODS: A Companion to Methods in Enzymology. 7, 147-161 (1995).</li><li>Sambrook, J., Russell, D. 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> Molecular Cloning - A laboratory manual. , (2001).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvABC+is+required+to+resolve+holliday+junctions+that+accumulate+following+replication+on+damaged+templates+in+Escherichia+coli.">RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli.</a> J Biol Chem. 281, 28811-28821 (2006).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvAB+and+RecG+Are+Not+Essential+for+the+Recovery+of+DNA+Synthesis+Following+UV-Induced+DNA+Damage+in+Escherichia+coli.">RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli.</a> Genetics. 166, 1631-1640 (2004).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecO+Acts+with+RecF+and+RecR+to+Protect+and+Maintain+Replication+Forks+Blocked+by+UV-induced+DNA+Damage+in+Escherichia+coli.">RecO Acts with RecF and RecR to Protect and Maintain Replication Forks Blocked by UV-induced DNA Damage in Escherichia coli.</a> J Biol Chem. 279, 3492-3496 (2004).</li><li>Belle, J. J., Casey, A., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+DnaB+helicase+leads+to+the+collapse+and+degradation+of+the+replication+fork:+a+comparison+to+UV-induced+arrest.">Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.</a> J Bacteriol. 189, 5452-5462 (2007).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecBCD+and+RecJ/RecQ+initiate+DNA+degradation+on+distinct+substrates+in+UV-irradiated+Escherichia+coli.">RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli.</a> Radiat Res. 168, 499-506 (2007).</li><li>Al-Hadid, Q., Ona, K., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecA433+cells+are+defective+in+recF-mediated+processing+of+disrupted+replication+forks+but+retain+recBCD-mediated+functions.">RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions.</a> Mutat Res. 645, 19-26 (2008).</li></ol>

图1显示了典型的从野生型细胞的存在和紫外线损伤的情况下取得的结果。 〜1％的总质粒DNA损害的情况下，可以发现，在Y弧，当细胞迅速增长指数相。照射后，在Y形分子的瞬时增加观察堵塞的复制叉积累损坏的网站。 X形复制的中​​间体，也瞬时积累和坚持，直到一段时间，当病变修复相关。 南分析，可以砸出复制中间体的凝胶，用塑料吸管，纯化，并直接通过电子显微?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>An example of the Southern analysis of the 2D gel probed</td>
<td>&nbsp;</td>
<td>GE</td>
<td>G15T8</td>
<td>Yellow Lighting</td>
</tr>
<tr>
<td>15 &mu;watt germicidal lamp</td>
<td>&nbsp;</td>
<td>Sylvania</td>
<td>F20T12/GO</td>
<td>UV Lamp</td>
</tr>
<tr>
<td>Blak-Ray UV Intensity Meter 254nm</td>
<td>&nbsp;</td>
<td>Daigger</td>
<td>EF28195T</td>
<td>UVC photometer</td>
</tr>
<tr>
<td>0.025 &mu;m pore disks</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>VSWP04700</td>
<td>Floating dialysis disks</td>
</tr>
<tr>
<td>PvuII</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>ER0632</td>
<td>Restriction Endonuclease</td>
</tr>
<tr>
<td>Nick-translation kit</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>976776</td>
<td>To make 32P-labeled probe</td>
</tr>
<tr>
<td>Blotting Paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-704</td>
<td>For Southern transfer</td>
</tr>
<tr>
<td>Nylon membrane</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>RPN203S</td>
<td>For Southern transfer</td>
</tr>		</tbody>
		</table>
		</div>

visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

在DNA损伤的存在不准确的复制是负责大部分的细胞重排，并观察在所有类型的细胞，是人们普遍认为，直接与人类的癌症发展相关的突变。如紫外线照射引起的的DNA损伤，严重损害了精确重复的基因组模板的复制能力。已经确定的基因产物，需要复制时，在模板中遇到的DNA损伤。然而，剩下的挑战已确定在复制过程中如何将这些蛋白质的过程中病变<em&gt;在体内</em&gt;。利用大肠杆菌作为模型系统，我们描述了二维琼脂糖凝胶电泳分析程序，可以用来识别复制的质粒上出现的结构性中间体<em&gt;在体内</em&gt;下面的紫外线引起的DNA损伤。使用这个程序已经证明，阻止紫外线损伤的复制叉经过一个短暂的逆转，是由RecA和几个基因相关产品与RecF通路稳定。这些复制中间体保持，直到时间的病灶切除核苷酸切除修复和复制恢复相关的技术演示。

Watch this Scientific Journal Video about Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis at JoVE.com

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

我们目前可以使用其中的一个两维的琼脂糖凝胶电泳分析，以确定紫外线照射后发生的复制中间体的结构的过程。

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

visualization-uv-induced-replication-intermediates-e-coli-using-two

Research

JoVE Journal

Biology

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

10.6K 次观看.  Portland State University. 我们目前可以使用其中的一个两维的琼脂糖凝胶电泳分析，以确定紫外线照射后发生的复制中间体的结构的过程。

视频: Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

科研

生物学

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

琼脂糖凝胶电泳分离DNA片段

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达<em&gt;电子。大肠杆菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

人类诺沃克病毒凸，域的生产<em&gt;电子。大肠杆菌</em&gt; X射线晶体学

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

粘蛋白琼脂糖凝胶电泳：蛋白质印迹高分子量糖蛋白


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...