1. Lysate 통관 <ol><li> E. 그로 37 밤새 적절한 항생제로 Luria의 수프에 깊은 우물을 접시에 원하는 플라스미드 DNA (문화 / 잘 ​​1 ML) · 잡고 C로 변형 대장균 문화. </li><li> 10 분 5,000 XG에 문화 접시에있는 펠렛 E. 대장균, 다음 가만히 따르다 뜨는. </li><li> 100 μL Resuspension 버퍼 (50 MM 트리스 - HCL, pH를 8.0, 10 MM EDTA (에틸렌 다이아 민 테트라 초산), 100 μg / ML RNase A)의 각 펠렛을 Resuspend. </li><li> 100 μL / 잘 용해 버퍼 (200 MM NaOH, 1 % SDS) 추가하고 2 분 동안 접시 통을 사용하여 발라줍니다. </li><li> 100 자 / μL 중화 버퍼 (3.0 M의 칼륨 아세테이트, 산도 5.5) 추가하고 2 분 흔들. </li><li> 설명 판을 lysate로 세포 lysate을 전송합니다. </li><li> 350 μL 수집 플레이트 및 진공 매니폴드의 하단에 자리 위에 플레이스 DNA 바인딩 플레이트. (그림 1 참조) 진공 매니폴드를 조립. </li><li> 진공 장치 위에 플레이스 lysate의 명확 플레이트. </li><li> HG (0.34 바) 인치 10 진공을 적용하고 DNA 바인딩 플레이트에 여과물를 수집합니다. HG 인치 12보다 큰 진공 청소기를 사용하지 마십시오. (0.41 바). </li><li> 진공 매니폴드를 분해. 장치의 아래쪽에 넣어 두 ML 폐기물 수집 플레이트. </li></ol> 2. DNA 바인딩 <ol><li> (그림 2 참조) 다기관의 맨 ​​위에 DNA 바인딩 플레이트를 이동합니다. </li><li> 300 μL / 잘 바인딩 버퍼 (6 M 구아니딘 - HCL)에 추가하고 혼합하고 아래로 피펫. </li><li> 진공을 적용 [~ 느린 진공에 대한 HG 인치 5 (0.17 바)]와 여과물을 삭제. DNA는 이제 막에 바인딩됩니다. </li><li> 와시 버퍼 (80 % 에탄올)의 / 음 400 μL로 씻으십시오. </li><li> 진공을 적용 후 여과액 폐기하십시오. 2 ML 수집 플레이트를 사용하거나 낭비 직접 필터링하는 경우 불필요 폐기하십시오. </li><li> 한번 세차를 반복합니다. </li><li> 건조 흡수 타월에 필터 플레이트의 하단에 얼룩. </li><li> 잔류 알코올의 제거를 위해 5-10분 위해 한번 더 진공 적용됩니다. </li><li> 에탄올 방울의 제거를 위해 바닥 얼룩. </li></ol> 3. DNA의 용출 <ol><li> 70 자 / μL 용출 버퍼 (10 MM 트리스 - HCL, pH를 8.0, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산)) 추가합니다. 높은 DNA 농도가 원하는 경우, 볼륨은 50 μL / 음에 줄일 수 있지만, 평균 총 수확량이 낮은 것입니다. </li><li> 일분 실온에서 접시를 품어. </li><li> 정화의 DNA는 진공 또는 원심 분리에 의해 eluted 수 있습니다. </li><li> 진공 방식 : 진공 매니폴드에 플레이스 깨끗한 수집 판 (DNase, RNase 무료). 모든 용출 버퍼 DNA 바인딩 플레이트 통과 때까지 진공 매니폴드 위에 플레이스 필터 플레이트는 1 분 HG (0.5 바) 인치 15 진공을 적용합니다. 정화의 DNA를 (그림 3 참조)를 수집합니다. </li><li> 원심 분리 방법 : 5 분 동안 1,000 XG에 깨끗한 수집 판과 원심 분리기 위에 플레이스 정화 필터 플레이트. </li></ol> 4. 양적 및 질적 분석 <ol><li> OD 280분의 260 측정, DNA를 정화의 농도를 계산합니다. </li><li> 정화 pDNA의 아가로 오스 겔의 분석을 수행합니다. </li></ol> 5. 대표 결과 E. 대장균이 자료를 시작 파편 감지할 수있을 정도의 감소를 보여 기존의 원심 분리와 팔 AcroPrep 사전 lysate의 명확 필터 플레이트를 사용하여 명확히 lysate. (그림 4). 세 농도에서 플라스미드 DNA는 TE 버퍼로 아군과 AcroPrep 사전 lysate의 명확 필터 플레이트 통과되었다. 플라스미드 DNA 농도의 비교, 사전 및 사후 여과, 쇼는 세 가지 농도 100 % 복구 (그림 5) ~. 명확히 E.에서 DNA의 복구를 입증하기 위해 대장균 lysate, 플라스미드 DNA가 원유 lysate 치솟았어 후 여과하여 명확히했다. 동일한 플라스미드 DNA가 세포 lysate의 다음 여과에 추가되었습니다. 전기는 각 샘플 (그림 6)에서 aliquots에서 수행되었다. 각 샘플에서 유사한 강도의 하나, 명확 밴드는 여과 기반 설명의 단계에서 작은 경우에는 모든 DNA 손실을 보여줍니다. E.의 문화 pCAT 플라스미드 DNA를 포함하는 대장균은 E.에서 정화되었습니다 대장균은 팔의 350 μL 1 ML의 DNA 바인딩 필터 플레이트뿐만 아니라, 350 μL DNA 바인딩 접시 두 가지 다른 브랜드를 사용 lysates. 제조 업체에서 권장하는 프로토콜에 명시된 바와 같이 모든 purifications이 수행되었다. 가장 높은 DNA 농도와 총 생산량은 팔의 1 ML AcroPrep 사전 DNA 정화 필터 플레이트 (그림 7과 표 2)에서 볼 수 있습니다. 경쟁 W 또한 높은 DNA 농도를 (155 NG / μL)를 보여줍 있지만, 전체 DNA의 수율이 (4.7 μg / 음) 낮은 복구 볼륨으로 인해 가장 낮은이며, ~ 30 μL. 경쟁 M 108에서 DNA의 가장 낮은 농도를 준 5.6 μg / 음의 총 수율과 μL / NG. 한천세 가지 DNA 바인딩 접시에서 pCAT DNA를 정화의 오세 겔 분석 그림 8에 표시됩니다. 각 수집된 샘플 볼륨이 전기하기 전에 복구 볼륨의 차이에 대한 정확한 65 μL로 조정되었습니다. 모든 샘플은 플라스미드 DNA를 supercoiled 보여주지만, 필터 플레이트의 각 DNA의 수율이 달라집니다. 그림 9는 준비 DNA가 supercoiled 양식에 모두 있으며, 유사한 농도를 가지고 보여줍니다. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="그림 1" /> 그림 1. Lysate 자세한 내용을 진공 매니폴드의 조립. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="그림 2" /> 그림 2. DNA 바인딩에 대한 진공 매니폴드의 조립. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="그림 3" /> 그림 3. 진공 용출을위한 진공 매니폴드의 조립. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="그림 4" /> 그림 4. 여과는 샘플 허가를 단순화합니다. 진공 여과 (350 μL AcroPrep 사전 lysate의 명확 필터 플레이트) 또는 전통적인 원심 분리에 의해 명확히 원유 lysate 300 μL의 세중의 샘플. OD 320 설명 전후에 측정. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="그림 5" /> 그림 5. AcroPrep 사전 lysate의 명확 필터 플레이트를 통해 통과 후 pDNA의 전체 복구. 300 μL TE 버퍼는 25, 50 및 100 μg / ML에서 pCAT를 타서. DNA 농도 및 복구, N = 2 전, 여과 후 OD 260에서 계산. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="그림 6" /> 그림 6. 설명 후 pCAT DNA의 복구. 이전이나 이후에 설명 lysate 치솟았어 pUC19 동등한 금액은 다음 TE 버퍼 10에 1을 희석. 1.2 % 아가로 오스 겔 2 μL / 레인로드됩니다. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="그림 7" /> 그림 7. 팔 Acroprep 사전 DNA 경쟁 플레이트보다 구속력이 필터 플레이트 잘마다 높은 플라스미드 DNA를 얻을 수 있습니다. 1 ML (팔와 경쟁 업체의 M) 1.5 ML (경쟁 W)의 야간 문화 표시 DNA 정화 플레이트를 사용하여 pCAT 플라스미드 DNA의 수율 (OD 260) DH5α. 강판 제조 업체의 권장 프로토콜을 사용하여 정제. 오차 막대는 표준 오류 (N&gt; 6) 나타냅니다. <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="그림 8" /> 그림 8. pCAT의 DNA의 아가로 오스 겔 분석 표시된 DNA 바인딩 접시에 정화. 정화 pDNA의 1.2 % 아가로 오스 겔 전기 영동. 세 가지 purifications에서 풀링된 샘플은 eluate는 정화, 희석 1시 10분 후 65 μL 조정. 레인 당 2 μL 일만 했어. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="그림 9" /> 그림 9. 품질 및 원심 분리기 또는 진공 방법을 정화 pDNA 최종 eluate 수집 아가로 오스 겔 전기 영동에 사용하는 경우와 비슷한 순수한 DNA의 복구. AcroPrep 사전 필터 플레이트 제조 업체에서 권장하는 프로토콜은 pDNA 정화에 대한 미행했다. 용리 2 분 HG (0.5 바) 인치 15 오분이나 진공 1,000 XG에서 원심 분리기에 의해 수행되었다.

이 비디오 문서의 저자는 악기와 비디오 및 텍스트에 등장하는 시약을 생산 팔 생명 과학의 직원입니다.

우리는 몇 가지 장점을 내실수 AcroPrep 사전 필터 플레이트의 손쉬운, 간소화된 프로토콜을 설명합니다. 수동 세미 자동, 또는 완전 자동 AcroPrep 사전 형식으로 처리하면이 방법은 최대 플라스미드 DNA의 수율을 제공합니다. lysate 통관 접시에 통합 prefilter도 샘플, 입자의를 포함 높은 수준의 일관성있는 여과를 제공합니다. 보다 빠르고 균일 최소한의 개최 - 최대 볼륨으로 접시에 걸쳐 여과 속도에 판 결과 잘 도형 및 아울렛 팁 기하학은 교차 오염의 우려없이 수신기 플레이트에 샘플을 직접 흐름을 제공합니다. 중요한 AcroPrep 사전 필터 플레이트의 DNA 결합 판의 높은 바인딩 용량 막은 높은 품질의 플라스미드 DNA의 최대 회복에 최적입니다.

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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>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

우리는 prokaryotic 문화에서 시작하고 고순도 DNA로 끝나는, 96 플라스미드 준비를위한 AcroPrep 사전 필터 플레이트의 전체 과정을 설명합니다. 세균성 lysate 통관 및 DNA의 정화를위한 멀티 잘 여과에 따라이 방법은 플라스미드 준비를위한 능률적인 프로세스를 만듭니다. 실리카 기반의 미디어를 포함하는 필터 플레이트는 쉽게 진공 여과 또는 플라스미드 DNA가 감지할 수있을 정도의 수량을 항복하기 위해 원심 분리기에 의해 처리할 수 있습니다. 정량 분석은 정화 플라스미드 DNA가 1.97의 평균 OD 280분의 260 비율로 양질의 지속 결정. 전반적으로, 플라스미드 수율은 시퀀싱 및 복제와 같은 다운 스트림 응용 프로그램에 대한 더 순수한 DNA를 제공합니다. AcroPrep 사전 필터 번호판을 사용하는 이것은 능률적인 방법은, 수동 세미 자동 또는 완전 자동으로 처리 수 있습니다.

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

이 프로토콜은 효율적으로 병렬에서 설명 및 플라스미드 DNA의 정화를위한 비용 효율적인 대안입니다<em&gt; E. 대장균</em&gt; 문화. AcroPrep 사전 프로세스가 높은 바인딩 용량 DNA 바인딩 필터를 접시에 정화 다음 최적의 lysate의 명확 필터 플레이트와 함께 시작합니다.

Prokaryotic 문화 플라스미드 DNA의 유선형 정화

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

streamlined-purification-of-plasmid-dna-from-prokaryotic-cultures

Nanyang Technological University

Research

JoVE Journal

Biology

19.9K 조회수.  Pall Life Sciences.  中文 

비디오: 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.

Qiagen Miniprep 키트를 사용하여 세균의 플라스미드 식민지에서 DNA를 정화

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

연구

생물학

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.

열 충격 방법을 사용 E. 대장균에 플라스미드 DNA의 변화

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.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

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.

효모 세포 Mitochondria의 정화

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.

에서 기본 셀 문화 Drosophila Gastrula의 태아

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.

골격근에서 Progenitors의 정화

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.

Kinesin의 분리 및 정제에서 Drosophila 태아

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.

이전 DNA의 정제없이 식물과 동물 샘플의 Genotyping

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 / 카스 시스템의 Endonucleases을위한 기판 생성

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.

성적 증명서와 Metabolites에서의 정화<em&gt; Drosophila</em&gt; 헤드

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