1。裂解液间隙<ol><li>成长大肠杆菌改造所需的质粒DNA在深孔板（1毫升文化/）卢里亚肉汤与适当的抗生素一夜之间在37℃用颤抖的彗星的大肠杆菌培养。 </li><li>佩莱大肠杆菌的培养板在5000 10分钟的XG，然后倒出上清液。 </li><li>每100μL，再悬浮缓冲液（50毫米的Tris - HCl，pH值8.0，10 mM的EDTA，100微克/毫升RNA酶A）重悬沉淀。 </li><li>加入100μL/孔裂解液（200毫米氢氧化钠，1％SDS）和摇板振动筛2分钟。 </li><li>加入100μL/孔和缓冲液（3.0 M醋酸钾，pH值5.5），振摇2分钟。 </li><li>转移细胞裂解液，裂解液澄清板。 </li><li> DNA结合的板350μL收集板和成真空歧管底部的地方上。组装真空歧管（见图1）。 </li><li>裂解液放置澄清真空装置上板。 </li><li>应用在10英寸汞柱（0.34巴）真空，滤液收集到的DNA结合板。不要使用大于12英寸汞柱真空。 （0.41 bar）的。 </li><li>拆开真空歧管。将2毫升废物收集板装置的底部。 </li></ol> 2。 DNA结合<ol><li>移动的DNA结合板多方面的顶部（见图2）。 </li><li>加入300μL/孔约束力的缓冲区（6 M盐酸胍）和吸管向上和向下混合。 </li><li>应用真空〜5英寸汞柱（0.17巴）缓慢真空和丢弃滤液。 DNA是现在势必膜。 </li><li>与400μL洗涤缓冲液（80％乙醇）/清洗。 </li><li>应用真空，然后丢弃滤液。如果使用2毫升收集板或过滤直接浪费丢弃不必要的。 </li><li>重复洗一次。 </li><li>吸干底部过滤板吸水毛巾擦干。 </li><li>应用真空一次5-10分钟，以确保去除残留的异丙醇。 </li><li>吸干底部，以确保去除乙醇滴。 </li></ol> 3。 DNA的洗脱<ol><li>添加70μL/孔洗脱缓冲液（10毫米的Tris - HCl，pH值8.0，1毫米EDTA）。如果需要较高的DNA浓度，体积可减少到50μL/孔，但平均总收益率将降低。 </li><li>一分钟，在室温下孵育板。 </li><li>可无论是真空或离心洗脱纯化的DNA。 </li><li>真空法:将清洁的收集板（脱氧核糖核酸酶，核糖核酸酶自由）进入真空系统。放在真空歧管上的过滤板，适用于15英寸汞柱（0.5巴）1分钟真空，直到所有的洗脱缓冲液通过DNA结合板通过。收集纯化后的DNA（见图3）。 </li><li>离心法:将清洁收集板的顶部和1000 XG 5分钟离心净化过滤板。 </li></ol> 4。定量和定性分析<ol><li>测量外径260/280，计算纯化的DNA的浓度。 </li><li>进行了纯化的质粒DNA琼脂糖凝胶电泳分析。 </li></ol> 5。代表性的成果 大肠杆菌裂解液澄清使用传统的离心和帕尔AcroPrep提前裂解液澄清过滤板显示的碎片明显减少，从起始原料。 （图4）。 到TE缓冲液中加入三个浓度的质粒DNA，并通过AcroPrep提前裂解液澄清过滤板通过。质粒DNA浓度的比较，前，后过滤，显示在所有三个浓度〜100％的恢复（图5）。 为了证明澄清大肠杆菌的DNA恢复添加到原油裂解大肠杆菌裂解，质粒DNA，然后通过过滤澄清。相同的质粒DNA加入到细胞裂解液以下过滤。电泳是从每个样本（图6）等分。一个单一的，从每个样品类似强度的明确乐队演示一点在澄清过滤步骤如有DNA损失。 文化的E.从 E 大肠杆菌含有PCAT质粒DNA纯化大肠杆菌裂解液使用颇尔公司的350μL和1毫升的DNA结合过滤板，以及两个其他品牌的350μL的DNA结合板。所有纯化制造商建议的协议规定执行。颇尔公司的1毫升AcroPrep进展DNA纯化过滤板（图7和表2）从最高的DNA浓度和总收率。 虽然竞争对手W也显示了DNA浓度高（155毫微克/μL），总DNA的产量是最低的（4.7微克/孔），由于回收量低，〜30微升。竞争者中号了108纳克/μL总产的5.6微克/以及DNA的最低浓度。 洋菜OSE凝胶纯化从三种不同的DNA结合板PCAT DNA分析，如图8所示。每次采集的样本量调整至65μL，以正确的回收量的差异，以电泳前。所有样品超螺旋质粒DNA，但每个滤板的DNA收益率各不相同。 图9显示了所有准备的DNA超螺旋的形式，也有类似的的浓度。 <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="图1" /> 图1。大会歧管的真空裂解液澄清 。 <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="图2" /> 图2。大会的DNA结合真空歧管 。 <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="图3" /> 图3。大会真空歧管真空洗脱。 <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="图4" /> 图4。过滤简化了样品澄清。一式三份样品300μL真空过滤（350微升AcroPrep提前裂解液澄清过滤板）或传统的离心澄清原油裂解。外径320测量之前和之后的澄清。 <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="图5" /> 图5。 PDNA通过后，通过AcroPrep提前裂解液澄清过滤板的全面恢复。 300μLTE缓冲液中添加与PCAT在25，50和100微克/毫升。 DNA浓度和恢复计算外径260过滤前后，N = 2。 <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="图6" /> 图6。 PCAT的DNA后的恢复澄清 。裂解液之前或之后澄清飙升的pUC19相等，则稀释1至10日在TE缓冲液。装2微升/ 1.2％琼脂糖凝胶上的车道。 <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="图7" /> 图7。质粒DNA，每口井产量与PALL Acroprep进展DNA结合比竞争对手板过滤板 。PCAT质粒DNA产量（ 外径 260）使用1毫升（帕尔和竞争对手中号）或1.5毫升（竞争对手宽）过夜培养的指示的DNA纯化板DH5α中。净化用板制造商的推荐协议。误差线表示标准错误（N&gt; 6）。 <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="图8" /> 图8。琼脂糖凝胶电泳分析PCAT的DNA纯化表示DNA结合板 。 1.2％，纯化的质粒DNA琼脂糖凝胶电泳。由三个独立的净化池的样品，洗脱液净化后，稀释1:10调整至65μL。装2％μL巷。 <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="图9" /> 图9。纯DNA相似的离心或真空方法最终洗脱液收集纯化的质粒DNA琼脂糖凝胶电泳时的质量和恢复。其次PDNA净化AcroPrep预先过滤板制造商的建议协议。洗脱在1000 XG离心5分钟或2分钟与15英寸汞柱（0.5巴）的真空。

此视频文章的作者是谁生产仪器和试剂的视频和文字的特色，颇尔生命科学的员工。

我们描述了一个浅显的，精简AcroPrep提前滤板的协议，给予几个优点。在手工，半自动化或全自动化AcroPrep提前格式的处理时，这种方法提供了最大的质粒DNA产量。对裂解间隙板的集成预过滤，提供样品，即使是那些含有较高水平的微粒过滤一致。更快，更均匀，整个板块的滤过率，以最小的滞留量板的结果以及几何;和出口尖几何提供了直接到接收板样品流，无交叉污染的关注。更重要的是，高的DNA结合能力AcroPrep预先过滤板具有约束力板膜是优化高质量的质粒DNA的最大恢复。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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>AcroPrep Advance Filter plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8175 (1 mL) or PN 8075 (350 &mu;l)</td>
<td>3 &mu;m glass fiber 0.2 &mu;m Supor&reg; membrane for clarification</td>
</tr>
<tr>
<td>AcroPrep Advance Filter Plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8132</td>
<td>for DNA Binding</td>
</tr>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 5017</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pressure pump</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 13157</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

streamlined purification of plasmid dna from prokaryotic cultures

我们描述AcroPrep提前96个质粒的准备滤板的完整的过程，从原核文化，并以高纯度的DNA结尾。基于多井过滤细菌裂解液检查和DNA纯化，这种方法创建一个质粒编制的精简过程。含有硅为基础的媒体的过滤板可以很容易地处理，真空过滤或离心产生可观数量的质粒DNA。定量分析确定纯化的质粒DNA是高品质的一贯平均OD 260/280比值1.97 。总体而言，质粒产量提供更纯净的DNA测序和克隆，下游应用，如。这种简化方法采用AcroPrep提前滤板允许手动，半自动或全自动化处理。

Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

此协议是一个成本效益的替代方案，为高效的并行澄清和质粒DNA纯化<em&gt; E。大肠杆菌</em&gt;文化。 AcroPrep发展的过程开始于一个优化的裂解液澄清过滤净化结合能力高的DNA结合滤板的车牌。

从原核文化精简纯化的质粒DNA

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

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JoVE Journal

Biology

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视频: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

科研

生物学

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 ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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.

基因分型的植物和动物样品，恕不另行DNA的纯化

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...