Die Arbeit in Dr. Bermudez Labor wurde von CONACYT (Grant # 82622) gefördert. Wir danken Gabriela Gutierrez-Sosa, Eliuth Reyes-Martinez und Ximena Rodriguez-Torres für die Videoaufzeichnung der electroleution Verfahren.

<ol><li> Um DNA-Fragmente von Interesse zu reinigenden wählen, sollten diese in Agarose aufgelöst werden oder Acrylamidgelen Gel mit Ethidiumbromid durch Elektrophorese mit 0,5 X TBE-Puffer (Tris-Borat, EDTA) bei 120 Volt für 1 Stunde gefärbt. Dafür ~ 700 ng Plasmid pProEx-GdMre11S (6000-bp), die zuvor mit NdeI und HindIII verdaut wurde ein 900-bp-Fragment (560 ng Plasmid und 84 ng des Fragments) freizugeben. </li><li> Die electroeluter Tank (Abbildung 1) sollte mit 0,5 X TBE gleichermaßen in jeder Seite der Mitte Plateau verteilt ausgefüllt werden betreut nicht verschütten es auf die Mitte der Hochebene. </li><li> Die ausgewählten Bands (oder Bands) aus dem Gel ausgeschnitten, und in Probenkammer so nah wie möglich an den V-Kanal (eine Scheibe pro Well). Laufpuffer sollte jeder Kammer hinzugefügt werden; gerade genug, um das Gel Scheibe abdecken. </li><li> Jeder V-Kanal verwendet werden, müssen mit einer Pasteurpipette gespült werden, um jede Luftblase gebildet zu beseitigen. </li><li> Dann wurden 100 ul von 10 M NH 4-Acetat (zur Visualisierung erleichtert eine kleine Menge von Bromphenolblau hinzugefügt wird, gerade genug, um es Farbe) werden sanft in die V-Kanal hinzugefügt. </li><li> Der Deckel sollte sanft geschlossen werden keine Aufwirbelung des hohen Salz-Kissen zu verhindern. </li><li> Elektroelution ist bei 100 Volt für 25 Minuten begonnen. </li><li> Wenn electrolution abgeschlossen ist, werden 400 ul von V-Kanal entfernt, und in ein Mikrozentrifugenröhrchen mit 2 Volumina kaltem Ethanol und Glykogen (empfohlen DNA-Ausbeute zu erhöhen) bei 4 ° C für 1 Stunde oder über Nacht ausgefällt werden. </li><li> Zentrifuge für 20 Minuten, entfernen Sie den Überstand und wäscht mit 70% Ethanol. </li><li> Dry Rohr und Quantifizierung der DNA bei OD 260 nm. </li><li> Die Erholung erhalten wurde, 75% (63 ng erholt Wenn 84 ng auf dem V-Kanal gelegt wurden). </li></ol><img src="/files/ftp_upload/2136/2136fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Electroeluter Diagramm. Anod ein Kathode auf jeder Seite des Tanks, DNA im Gel geschnitten enthalten sind, angegeben (dargestellt als ein schwarzes Quadrat) wird in Richtung Kathode durch ihre negative Ladung zu migrieren. Dann wird es in das Salz Kissen (dargestellt als ein umgekehrtes schwarzes Dreieck) in der V-Kanal befindet sich gefangen zu sein.

<ol>
	<li>Sambrook, J., Fritsch, E. F., Maniatis, T. <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. , (1989).</li></ol>

Die Dauer des Laufs wird in hohem Maße abhängig von der Größe der Fragmente, die normalerweise für Fragmente bis zu 20 kb 50 Minuten bis 1 Stunde ist genug, während für kleine Fragmente UV-Licht Überwachung alle 10 Minuten ist erforderlich, um das Fragment zu verhindern, um durch die go Salz Kissen und damit auf die anodische Pufferkammer abnehmender Erholung. Es ist wichtig, um sicherzustellen, dass, wenn Sie Ihr Netzteil, eingestellt auf 100 Volt, gibt es eine aktuelle mindestens von 10 MAMP. Die DNA-Bande kann durch Verwendung eines UV-Handlampe überwacht werden und erkennen, wenn diese verlässt das Gel in Scheiben schneiden. Das Polster Salz kann auch mit 3 M Na-Acetat werden. Dieses Verfahren ermöglicht die Aufreinigung von DNA oder RNA-Fragmente unterschiedlicher Größe mit einer guten Erholung (~ 80%) auf radioaktiv werden, verdaut durch Restriktionsenzyme, verarbeitet durch modifizierende Enzyme, etc.

<table><tbody><tr><td>NdeI</td><td>New England Biolabs</td><td>R0111L</td><td></td></tr><tr><td>HindIII</td><td>New England Biolabs</td><td>R0104L</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt; acetate</td><td>JT Baker</td><td>0596</td><td></td></tr><tr><td>agarose</td><td>Invitrogen</td><td>15510-027</td><td></td></tr><tr><td>Biophotometer</td><td>Eppendorf</td><td>6131 000 020</td><td></td></tr><tr><td>Electr&amp;#8211;luter</td><td>IBI</td><td>UEA CAT 46000</td><td></td></tr></tbody></table>

electroeluting dna fragments

Gereinigte DNA-Fragmente werden für verschiedene Zwecke in Molecular Biology eingesetzt und sie können durch mehrere Verfahren hergestellt werden. Die meisten von ihnen erfordern eine vorherige Elektrophorese der DNA-Fragmente, um die Band von Interesse zu trennen. Dann wird dieses Band aus einem Agarose oder Acrylamid Gel ausgeschnitten und gereinigt, indem Sie entweder: Bindung und Elution aus Glas oder Silica-Partikel, DEAE-Cellulose-Membranen, &quot;crush und genießen Methode&quot;, Elektroelution oder sehr oft teuren kommerziellen Aufreinigungskits. So wird die Auswahl einer Methode hängt vor allem von dem, was im Labor zur Verfügung. Die Elektroelution Verfahren erlaubt es, sehr sauber DNA in einer Vielzahl von Anwendungen (Sequenzierung, Radiomarkierung, enzymatische Restriktion, enzymatische Modifikation, Klonen usw.) verwendet werden zu reinigen. Dieses Verfahren besteht in Platzierung DNA-Bande enthaltende Agarose oder Acrylamid Scheiben in Probenvertiefungen der electroeluter, dann Anlegen von Strom wird das DNA-Fragment auf die Agarose zu verlassen und damit in ein Kissen Salz gefangen werden, um später durch Ethanolfällung gewonnen werden.

Watch this Scientific Journal Video about Electroeluting DNA Fragments at JoVE.com

Electroeluting DNA Fragments

Dieses Verfahren ermöglicht die Aufreinigung von DNA-Fragmenten mit hoher Ausbeute.

Electroeluting DNA-Fragmenten

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a ...

Nanyang Technological University

Research

JoVE Journal

Biology

27.7K Aufrufe.  Research and Advanced Studies Center of National Polytechnic Institute. Dieses Verfahren ermöglicht die Aufreinigung von DNA-Fragmenten mit hoher Ausbeute.

Serie: Electroeluting DNA Fragments

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Nachweis von Bakterien mit fluorogenen DNAzyme

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Forschung

Immunologie und Infektion

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

DNA-Extraktion von 0,22 pM Sterivex Filter und Cäsiumchlorid-Dichtegradientenzentrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Biologie

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extraktion von High Molecular Weight genomischer DNA aus Böden und Sedimenten

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

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Extraktion von DNA mit hohem Molekulargewicht von mikrobiellen Matten

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

<meta charset="utf8"></head><body>CMG-vermittelte DNA-Dewindung durch Wachstum des ssDNA-RPA-Trakts beobachtet

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Entschlüsselung der Dynamik der eukaryotischen DNA-Replikation durch Einzelmolekül-Visualisierung

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Einzelmolekül-Echtzeit-Visualisierung der DNA-Abwicklung durch CMG-Helikase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

Biochemie

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.

Agarosegel-Elektrophorese für die Trennung von DNA-Fragmenten

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 ...

DNA Isolation and Restriction Enzyme Analysis

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics.

<h4>Isolating DNA from Cells</h4>

DNA isolation is a fairly simple procedure that requires few but essential steps: cell harvesting, lysis, protein degradation, and finally DNA precipitation. Generally, DNA can be harvested from small amounts of tissue, such as a single leaf of a plant, hair follicles of animals, or epithelial cells that can be easily sloughed off within the mouth with a simple saline solution. Since DNA is compartmentalized inside the nucleus or mitochondria within eukaryotic cells, scientists first break apart the cell and nuclear membrane to expose the DNA, which is done with the lysis step. However, once DNA is exposed, it can be degraded by heavy metal ions and extremes of pH. Therefore, a detergent-containing buffer solution is used to not only dissolve the cell membrane, but also to protect DNA from degrading elements in the solution. Moreover, DNA is an unusually long molecule that is condensed inside the nucleus by packing tightly around special proteins called histones. Therefore, an enzyme called Proteinase K, which degrades peptide bonds of proteins, is added to separate the DNA from histones and other proteins. Next, scientists separate DNA from the rest of the cellular extract by using its chemical properties. Due to the negatively charged structure of nucleic acids, Na+ ions in a sodium chloride solution bind to and integrate into entangled DNA strands. Once the DNA is clumped, it can be observed by the naked eye. DNA is insoluble in alcohol and readily precipitates at low temperatures, thus scientists add cold alcohol to precipitate DNA. Finally, the precipitated DNA can be dissolved and stored in deionized water or a mild buffer, free of any enzymes that can degrade nucleic acids, until it is used.

<h4>Quantifying DNA</h4>

Reactions that use DNA often require high purity and precise amounts of DNA. Therefore, following DNA isolation, scientists use a spectrophotometer to quantify the amount of DNA in their sample, as well as the purity of the DNA in the sample. A spectrophotometer measures the intensity of a light beam passed through a sample as a function of its wavelength. Nucleic acids absorb light at 260 nm, whereas proteins absorb light at 280 nm. By measuring the intensity of a light beam at 260 nm, scientists can determine the DNA concentration. Furthermore, by assessing the light intensity at both 260 nm and 280 nm, the purity of the DNA content can be determined. In a sample of pure DNA, the 260/280 nm ratio should approach the value of 2.

<h4>Restriction Enzymes</h4>

For a number of analyses, such as cloning, sequencing, and mapping DNA fragments and genomes, the isolated DNA needs to be cut at specific sequences using restriction enzymes. These enzymes are produced by bacteria as mechanisms of innate immunity against intruding viral DNA and function as molecular scissors. Hence, after restriction enzymes identify a viral DNA, they start to break it apart by cutting it into segments at certain nucleotide sequences they recognize. These nucleotide sequences are often six to twelve nucleotides long and are generally palindromic, which means that they have the same nucleotide sequence in the 3&#8217;-5&#8217; and 5&#8217;-3&#8217; directions. For instance, the restriction enzyme EcoRI cuts DNA at the sequence 3&#8217;...GAATTC&#8230;-5&#8217;, which reads the same in the 5&#8217;-3&#8217; direction. Like EcoRI, there are hundreds of enzymes that correspond to many distinct palindromic sequences. Once DNA is cut by these enzymes, scientists can load the DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size. After the completion of the electrophoresis, DNA fragments can be visualized with the aid of dyes that specifically bind to DNA molecules.

Cutting DNA with restriction enzymes and separating DNA fragments with electrophoresis allows researchers to identify the sizes of unknown fragments relative to a standard ladder. These fragments of interest can then be further isolated and ligated into a plasmid vector for genetic cloning. Genetic engineering and laboratory analyses have been improved immensely with these methods. With DNA profiling, scientists are able to sequence genomes to further understand the workings of organisms and are also able to identify and characterize certain genes1.

DNA isolation and profiling are also practical for events directly impacting everyday life. For example, forensics scientists routinely analyze DNA samples to provide evidence for criminal and civil cases2. Similarly, healthcare workers extract and analyze DNA to check for paternity and genetic diseases. Recently, DNA ancestry analysis kits became popular by allowing users determine their genetic lineage, find relatives, and even discover their genetic disposition to various health conditions3. Finally, with advancements in biotechnology and genetic engineering, personalized medicine is gaining traction among clinical researchers to develop treatments by using an individual&#8217;s genetic data, paving the way to effectively treat patients without detrimental side effects4.

<h4>References</h4>

<ol class="references">
	<li>Chambers, GK, et al. DNA fingerprinting in zoology: past, present, future. Investig Genet. 2014, Vol. 5, 3 doi: 10.1186/2041-2223-5-3.</li>
	<li>Roewer, L. DNA fingerprinting in forensics: past, present, future. Investig Genet. . 2013, Vol. 4, 22 doi: 10.1186/2041-2223-4-22.</li>
	<li>Kirkpatrick, BE and Rashkin, MD. Ancestry Testing and the Practice of Genetic Counseling. J Gen Counseling. 2016, Vol. 26, 1 (6-20).</li>
	<li>Hamburg, MA and Collins, FS. The Path to Personalized Medicine. N Engl J Med . 2010; , Vol. 363, 301-304.</li>
</ol>

DNA-Isolierung und Restriktionsenzymanalyse

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our ...

Lehre

JoVE Lab Manual

Genetik

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of agarose, and within the gel, charge particles will migrate and separate based on size. The negatively charged phosphates of the DNA backbone cause DNA fragments to move toward the anode - a positively charged electrode.
The video explains the mechanism by which DNA fragments are resolved on an agarose gel, and it provides a step-by-step generalized procedure for how to prepare agarose gels, load DNA samples, run a DNA gel, visualize DNA fragments, and properly dispose of the gel and running buffer after the experiment is concluded.

DNA Gel Electrophoresis: Concept, Procedure, and Applications

DNA-Gelelektrophorese

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

Electroeluting DNA-Fragmenten

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Nachweis von Bakterien mit fluorogenen DNAzyme

DNA-Extraktion von 0,22 pM Sterivex Filter und Cäsiumchlorid-Dichtegradientenzentrifugation

Extraktion von High Molecular Weight genomischer DNA aus Böden und Sedimenten

Extraktion von DNA mit hohem Molekulargewicht von mikrobiellen Matten

Einzelmolekül-Echtzeit-Visualisierung der DNA-Abwicklung durch CMG-Helikase

Einzelmolekül-Echtzeit-Visualisierung der DNA-Abwicklung durch CMG-Helikase

Einzelmolekül-Echtzeit-Visualisierung der DNA-Abwicklung durch CMG-Helikase

Agarosegel-Elektrophorese für die Trennung von DNA-Fragmenten

DNA-Isolierung und Restriktionsenzymanalyse

DNA-Gelelektrophorese