1. Preparation of the Gel
<ol>
	<li>Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a w/v percentage solution. The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask.</li>
	<li>Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).</li>
	<li>Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved.</li>
	<li>Add ethidium bromide (EtBr) to a concentration of 0.5 &mu;g/ml. Alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 &mu;g/ml EtBr for 15-30 min, followed by destaining in running buffer for an equal length of time.</li>
</ol>
Note: EtBr is a suspected carcinogen and must be properly disposed of per institution regulations. Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the staining of DNA are available; however EtBr remains the most popular one due to its sensitivity and cost.
<ol start="5">
	<li>Allow the agarose to cool either on the benchtop or by incubation in a 65 &deg;C water bath. Failure to do so will warp the gel tray.</li>
	<li>Place the gel tray into the casting apparatus. Alternatively, one may also tape the open edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells.</li>
	<li>Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box. Alternatively, the gel can also be wrapped in plastic wrap and stored at 4 &deg;C until use (Fig. 1).</li>
</ol>

2. Setting up of Gel Apparatus and Separation of DNA Fragments
<ol>
	<li>Add loading dye to the DNA samples to be separated (Fig. 2). Gel loading dye is typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel.</li>
	<li>Program the power supply to desired voltage (1-5V/cm between electrodes).</li>
	<li>Add enough running buffer to cover the surface of the gel. It is important to use the same running buffer as the one used to prepare the gel.</li>
	<li>Attach the leads of the gel box to the power supply. Turn on the power supply and verify that both gel box and power supply are working.</li>
	<li>Remove the lid. Slowly and carefully load the DNA sample(s) into the gel (Fig. 3). An appropriate DNA size marker should always be loaded along with experimental samples.</li>
	<li>Replace the lid to the gel box. The cathode (black leads) should be closer the wells than the anode (red leads). Double check that the electrodes are plugged into the correct slots in the power supply.</li>
	<li>Turn on the power. Run the gel until the dye has migrated to an appropriate distance.</li>
</ol>
3. Observing Separated DNA fragments
<ol>
	<li>When electrophoresis has completed, turn off the power supply and remove the lid of the gel box.</li>
	<li>Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the gel tray on paper towels to absorb any extra running buffer.</li>
	<li>Remove the gel from the gel tray and expose the gel to uv light. This is most commonly done using a gel documentation system (Fig. 4). DNA bands should show up as orange fluorescent bands. Take a picture of the gel (Fig. 5).</li>
	<li>Properly dispose of the gel and running buffer per institution regulations.</li>
</ol>
4. Representative Results
Figure 5 represents a typical result after agarose gel electrophoresis of PCR products. After separation, the resulting DNA fragments are visible as clearly defined bands. The DNA standard or ladder should be separated to a degree that allows for the useful determination of the sizes of sample bands. In the example shown, DNA fragments of 765 bp, 880 bp and 1022 bp are separated on a 1.5% agarose gel along with a 2-log DNA ladder.
<img alt="Figure 1." src="/files/ftp_upload/3923/3923fig1.jpg" /> 
Figure 1. A solidified agarose gel after removal of the comb.
<img alt="Figure 2." src="/files/ftp_upload/3923/3923fig2.jpg" /> 
Figure 2. A student adding loading dye to her DNA samples.
<img alt="Figure 3." src="/files/ftp_upload/3923/3923fig3.jpg" /> 
Figure 3. A student loading the DNA sample into a well in the gel.
<img alt="Figure 4." src="/files/ftp_upload/3923/3923fig4.jpg" /> 
Figure 4. An example of a gel documentation system.
<img alt="Figure 5." src="/files/ftp_upload/3923/3923fig5.jpg" /> 
Figure 5. An image of a gel post electrophoresis. EtBr was added to the gel before electrophoresis to a final concentration of 0.5 &mu;g/ml, followed by separation at 100 V for 1 hour. The gel was exposed to uv light and the picture taken with a gel documentation system.

<ol>
	<li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. , (2001).</li><li>Kirkpatrick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+agarose+gel+properties.">Overview of agarose gel properties.</a> Electrophoresis of large DNA molecules: theory and applications. , 9-22 (1991).</li><li>Helling, R. B., Goodman, H. M., Boyer, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+endonuclease+R&#8226;EcoRI+fragments+of+DNA+from+lambdoid+bacteriophages+and+other+viruses+by+agarose-gel+electrophoresis.">Analysis of endonuclease R&#8226;EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis.</a> J. Virol. 14, 1235-1244 (1974).</li><li>Smith, S. B., Aldridge, P. K., Callis, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+individual+DNA+molecules+undergoing+gel+electrophoresis.">Observation of individual DNA molecules undergoing gel electrophoresis.</a> Science. 243, 203-206 (1989).</li><li>Aaji, C., Borst, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+electrophoresis+of+DNA.">The gel electrophoresis of DNA.</a> Biochim. Biophys. Acta. 269, 192-200 (1972).</li><li>Lai, E., Birren, B. W., Clark, S. M., Simon, M. I., Hood, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+field+gel+electrophoresis.">Pulsed field gel electrophoresis.</a> Biotechniques. 7, 34-42 (1989).</li><li>Devor, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IDT tutorial: gel electrophoresis. , (2010).</li><li>Serwer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gels:+properties+and+use+for+electrophoresis.">Agarose gels: properties and use for electrophoresis.</a> Electrophoresis. 4, 375-382 (1983).</li><li>Dea, I. C. M., McKinnon, A. A., Rees, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tertiary+and+quaternary+structure+and+aqueous+polysaccharide+systems+which+model+cell+wall+adhesion:+reversible+changes+in+conformation+and+association+if+agarose,+carrageenan+and+galactomannans.">Tertiary and quaternary structure and aqueous polysaccharide systems which model cell wall adhesion: reversible changes in conformation and association if agarose, carrageenan and galactomannans.</a> J. Mol. Biol. 68, 153-172 (1972).</li><li>Sharp, P. A., Sugden, B., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+restriction+endonuclease+activities+in+H.+parainfluenzae+using+analytical+agarose-ethidium+bromide+electrophoresis.">Detection of two restriction endonuclease activities in H. parainfluenzae using analytical agarose-ethidium bromide electrophoresis.</a> Biochemistry. 12, 3055-3063 (1973).</li></ol>

We have nothing to disclose.

Agarose gel electrophoresis has proven to be an efficient and effective way of separating nucleic acids. Agarose's high gel strength allows for the handling of low percentage gels for the separation of large DNA fragments. Molecular sieving is determined by the size of pores generated by the bundles of agarose7 in the gel matrix. In general, the higher the concentration of agarose, the smaller the pore size. Traditional agarose gels are most effective at the separation of DNA fragments between 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one will need to use pulse field gel electrophoresis6, which involves the application of alternating current from two different directions. In this way larger sized DNA fragments are separated by the speed at which they reorient themselves with the changes in current direction. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis. Unlike agarose gels, the polyacrylamide gel matrix is formed through a free radical driven chemical reaction. These thinner gels are of higher concentration, are run vertically and have better resolution. In modern DNA sequencing capillary electrophoresis is used, whereby capillary tubes are filled with a gel matrix. The use of capillary tubes allows for the application of high voltages, thereby enabling the separation of DNA fragments (and the determination of DNA sequence) quickly.
Agarose can be modified to create low melting agarose through hydroxyethylation. Low melting agarose is generally used when the isolation of separated DNA fragments is desired. Hydroxyethylation reduces the packing density of the agarose bundles, effectively reducing their pore size8. This means that a DNA fragment of the same size will take longer to move through a low melting agarose gel as opposed to a standard agarose gel. Because the bundles associate with one another through non-covalent interactions9, it is possible to re-melt an agarose gel after it has set.
EtBr is the most common reagent used to stain DNA in agarose gels10. When exposed to uv light, electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release of energy (light) as the electrons return to ground state. EtBr works by intercalating itself in the DNA molecule in a concentration dependent manner. This allows for an estimation of the amount of DNA in any particular DNA band based on its intensity. Because of its positive charge, the use of EtBr reduces the DNA migration rate by 15%. EtBr is a suspect mutagen and carcinogen, therefore one must exercise care when handling agarose gels containing it. In addition, EtBr is considered a hazardous waste and must be disposed of appropriately. Alternative stains for DNA in agarose gels include SYBR Gold, SYBR green, Crystal Violet and Methyl Blue. Of these, Methyl Blue and Crystal Violet do not require exposure of the gel to uv light for visualization of DNA bands, thereby reducing the probability of mutation if recovery of the DNA fragment from the gel is desired. However, their sensitivities are lower than that of EtBr. SYBR gold and SYBR green are both highly sensitive, uv dependent dyes with lower toxicity than EtBr, but they are considerably more expensive. Moreover, all of the alternative dyes either cannot be or do not work well when added directly to the gel, therefore the gel will have to be post stained after electrophoresis. Because of cost, ease of use, and sensitivity, EtBr still remains the dye of choice for many researchers. However, in certain situations, such as when hazardous waste disposal is difficult or when young students are performing an experiment, a less toxic dye may be preferred.
Loading dyes used in gel electrophoresis serve three major purposes. First they add density to the sample, allowing it to sink into the gel. Second, the dyes provide color and simplify the loading process. Finally, the dyes move at standard rates through the gel, allowing for the estimation of the distance that DNA fragments have migrated.
The exact sizes of separated DNA fragments can be determined by plotting the log of the molecular weight for the different bands of a DNA standard against the distance traveled by each band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can be compared against the unknown DNA samples. It is important to note that different forms of DNA move through the gel at different rates. Supercoiled plasmid DNA, because of its compact conformation, moves through the gel fastest, followed by a linear DNA fragment of the same size, with the open circular form traveling the slowest.
In conclusion, since the adoption of agarose gels in the 1970s for the separation of DNA, it has proven to be one of the most useful and versatile techniques in biological sciences research.

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Agarose I</td>
			<td>Amresco</td>
			<td>0710</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma</td>
			<td>B7901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma</td>
			<td>B8026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Sigma</td>
			<td>E7637</td>
			<td>Carcinogenic-needs to be disposed of as hazardous waste</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher</td>
			<td>BP2401-212</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>G33-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylene cyanol FF</td>
			<td>Sigma</td>
			<td>X4126</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Investigator/FX gel documentation system</td>
			<td>Fotodyne</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Owl Easycast B1 mini gel electrophoresis system</td>
			<td>Thermo Scientific</td>
			<td>B1-PTM</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EPS 301 power supply</td>
			<td>GE Healthcare</td>
			<td>18-1130-01</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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;

Watch this Scientific Journal Video about Agarose Gel Electrophoresis for the Separation of DNA Fragments at JoVE.com

Agarose Gel Electrophoresis for the Separation of DNA Fragments

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

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 ...

agarose-gel-electrophoresis-for-the-separation-of-dna-fragments

Research

JoVE Journal

Biology

761.1K ビュー.  University of California Los Angeles. アガロースゲル電気泳動を用いたDNA断片の分離のための基本的なプロトコルが記述されています。

ビデオ: Agarose Gel Electrophoresis for the Separation of DNA Fragments

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

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

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

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

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

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

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

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

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

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Electroeluting DNA Fragments

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.

This procedure allows the purification of DNA fragments with high yield.

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

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

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

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

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

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

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

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...