우리는 해안과 열린 바다 바닷물의 낮은 산소 지역에 지속적인 연구를 지원하기위한 혁신, 브리티시 컬럼비아 기술 개발 기금과 국립 과학 및 캐나다의 공학 연구 협의회 (NSERC)에 대한 캐나다 재단 감사드립니다. 도스는 NSERC, 킬람와 툴라 기초 미생물 다양성과 진화에 대한 투자 센터에서 장학금으로 지원했다.

<ol><li> 여과 장치의 설정 <ol><li> 여과 장치를 설정하려면, prefilter 주택 단위로 등급 GF / D 필터를 배치 핀셋을 사용합니다. 대충 손으로 주택을 조입니다. 엄지의 규칙으로, 오버 강화하지 않거나 장치의 펌핑 시작되면 누출됩니다. </li><li> 조심스럽게 그것을 만지지 않고 상자 속에 든 대형 유리병에 내부 튜브를 삽입하여 autoclaved 뚜껑과 연결된 튜브로 상자 속에 든 대형 유리병 뚜껑을 교체합니다. 필요한 경우, 상자 속에 든 대형 유리병에 튜브를 안내 핀셋을 사용합니다. </li><li> 연동 펌프를 통해 외부 튜빙을 전달합니다. </li><li> 모두 외부 튜브 (상자 속에 든 대형 유리병 당 즉 2 대)의 끝 부분에 prefilter 주택 장치를 연결합니다. 펜치와 연결을 조인다. </li></ol></li><li> 해수 여과 <ol><li> 해수를 필터링 시작하려면 펌프의 전원을 켜고 1 흐름 속도를 설정할 수 있습니다. 물이가 올바른 방향으로 나오지되도록보세요. </li><li> 물 500 ML은 prefilter 단위 통해 1 L의 비커에 자유롭게 흘러 수 있습니다. </li><li> 펌프를 끄고, 그리고 prefilter 설정의 자유로운 끝에 남자 luer 피팅에 Sterivex 필터를 첨부합니다. 손으로 prefilter 주택 snugly를 조이십시오. </li><li> prefilter 주택에서와 prefilter 주택 상자 속에 든 대형 유리병에 이르게 튜브 사이의 연결에서 살펴보십시오. 누수가 발생하면 펌프를 끄고 느슨하게하고 그에 따라 prefilter 주택을 retighten. 뒷면에있는 펌프를 켭니다. </li><li> 4 L의 flasks에 여과물를 수집합니다. 일단 그들은 깨끗한 상자 속에 든 대형 유리병에 flasks가 비어 가득 찼습니다. 각 필터를 통해 전달되는 볼륨을 참고하시기 바랍니다. 수집 여과액는 바이러스에 대한 여과 등 추가로 처리할 수 있습니다. </li><li> 물도이 carboys 달았어 때까지 계속 필터링. 4-5 H / 20 L의 상자 속에 든 대형 유리병 사이의 일반적인 여과 시간 범위는 있지만, 이것은 물 내에서 바이오 매스의 양을에 크게 의존합니다. </li><li> 여과가 완료되면 펌프를 끄고 Sterivex 필터를 분리합니다. </li></ol></li><li> 필터 저장 <ol><li> 필터를 저장하려면 먼저 30-60 ML의 주사기와 함께 남아있는 물을 추방. </li><li> 피펫을 사용하여 시약의 저장뿐만 아니라를 위해 필터의 공간 ~ 200 UL을 유지, 필터 1.8ml 용해 버퍼를 추가합니다. </li><li> Parafilm의 작은 조각으로 Sterivex 필터의 하단과 상단을 밀봉하고, 날짜와 샘플 식별과 필터를 라벨. </li><li> -80에서 미리 표시 50 ML 팔콘 튜브에 필터를 저장 ° C </li><li> 주택 단위에서 필터를 제거하고 용해 버퍼 2 ML를 포함하는 표시 50 ML 팰컨 튜브에 그들을 놓으십시오. -80에서 이들을 보관 ° C.를 </li></ol></li><li> 대청소 <ol><li> 최종 정리, 0.1 M HCL을 포함하는 플라스크에 상자 속에 든 대형 유리병 안에 튜브를 삽입 </li><li> 원래 상자 속에 든 대형 유리병에 4L 술병으로 이어질 튜빙을 직접. </li><li> 펌프 스위치를 원래의 상자 속에 든 대형 유리병에서 실행을 통해 흐름을 수집, 3 실행합니다. </li><li> 마지막으로, autoclaved 물을 튜빙을 씻어. </li></ol></li></ol> 대표 결과 : 최종 결과 두 Sterivix 필터 0.22μm와 2.7μm의 크기 범위에서 약 10 L의 해수에서 미생물 바이오 매스를 포함하는 각각 구성되어 있습니다. 이것은 용해 버퍼에 저장된 및 DNA 추출을위한 준비가되었습니다. 또한,이 절차를 수집하고 GF / D prefilters에, 2.7 μm의보다 큰 바이오 매스를 집중하고 있습니다. 이것은 또한 용해 버퍼에 저장된 및 DNA 추출을위한 준비가되었습니다. 추가 처리 단계를 촬영하는 경우, 흐름을 통해 0.22 μm의는뿐만 아니라 바이러스 분수를 수집 아래로 집중하실 수 있습니다.

The authors have nothing to disclose.

그것은 달리 장비를 부식 것이다 소금물의 느린 흔적을 제거하는 데 사용 후 젖은 수건으로 벤치와 펌프를 닦아 필수적입니다. 절차는 간단하지만, 시간 요구 사항이 필터링되는 예제에서 바이오 매스이나 먼지의 농도에 따라 광범위한 수 있습니다.

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		<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>Peristaltic pump</td>
<td>&nbsp;</td>
<td>Masterflex (Cole Palmer)</td>
<td>77913-20, 77200-62, 77200-04</td>
<td>&nbsp;</td>
<tr>
<td>Tygon LFL tubing</td>
<td>&nbsp;</td>
<td>Masterflex (Cole Palmer)</td>
<td>06429-24</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Hose clamps</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>0683204</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>1/4&#x201D; x 3/16&#x201D; pipe adaptors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>31702-20</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Polypropylene prefilter housing</td>
<td>&nbsp;</td>
<td>ADVANTEC</td>
<td>501200</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Metal luer fitting (Hose end to Male luer lock)</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>31507-27</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>2&#x201C; Magnets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For filtration apparatus</td>
<td>&nbsp;</td>
<tr>
<td>3 port carboy caps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>GV 0.22 &mu;m STERIVIX filters</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SVGV01R5</td>
<td>&nbsp;</td>
<tr>
<td>47 mm diameter 2.7 &mu;m GF/D filters</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1823047</td>
<td>&nbsp;</td>
<tr>
<td>Lysis buffer</td>
<td> Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3)</td>
</tr>
<tr>
<td>0.1 % HCl</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For post-filtration rinse</td>
</tr>
<tr>
<td>Autoclaved water</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For post-filtration rinse</td>
</tr>		</tbody>
		</table>
		</div>

large volume (20l+) filtration of coastal seawater samples

흐름은 하류 미생물 커뮤니티 해안 해양 물, 영양 및 추적 가스 분석의 수집과 함께 시작됩니다. 이 방법의 경우, 샘플은 사니치​​ 후미에 HMS 존 스트 리클 랜드 운영의 갑판에서 수집되었다. 물 칼럼에서 0.22μm와 직경 2.7μm 사이의 범위 미생물 바이오 매스의이 동영상은 문서 대형 볼륨 (≥ 20 L) 여과,. 두 20L 샘플은 네 머리 회전을 갖춘 단일 펌프 장치를 사용하여 동시에 필터링할 수 있습니다. 여과 연장 여행 분야에서 일을하거나, 즉시 당일 여행 돌아오면됩니다. 각 sterivex 필터 유닛 통과 물의 양을 기록하는 것이 중요합니다. 샘플링 여행 사이의 biofilm 형성을 방지하기 위해, 모든 여과 장비는 희석 HCL 및 탈이온수로 씻어서 바로 사용 후 autoclaved해야합니다. 이 절차는 약 5 시간 플러스 치우기 추가 시간이 소요됩니다.

Watch this Scientific Journal Video about Large Volume 20L+ Filtration of Coastal Seawater Samples at JoVE.com

Large Volume 20L+ Filtration of Coastal Seawater Samples

물 칼럼에서 0.22μm와 직경 2.7μm 사이의 범위 미생물 바이오 매스의이 동영상은 문서 대형 볼륨 (≥ 20 L) 여과,.

대량 연안 해수 샘플 (20L +) 여과

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

large-volume-20l-filtration-of-coastal-seawater-samples

Research

JoVE Journal

Biology

Large Volume (20L+) Filtration of Coastal Seawater Samples

11.7K 조회수.  University of British Columbia - UBC. 물 칼럼에서 0.22μm와 직경 2.7μm 사이의 범위 미생물 바이오 매스의이 동영상은 문서 대형 볼륨 (≥ 20 L) 여과,.

비디오: Large Volume 20L+ Filtration of Coastal Seawater Samples

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic 도서관 대규모 화면

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

연구

생물학

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

해수 샘플링 및 수집

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

연안 해수 샘플을 소량 (1 - 3L)은 여과

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

대형 삽입 환경 게놈 라이브러리 제작

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

대형 Unilamellar Vesicles로 펩타이드의 Translocation 측정

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

의식 쥐의 사구체 여과율의 측정을위한 높은 처리량 방법

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

조직 해리 차동 여과 이식 미토콘드리아의 신속한 분리 및 정제

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

생리학 실험실 데모 : 쥐의 사구체 여과 속도

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

마우스에 압력 볼륨 루프 측정

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...