Name: electroeluting dna fragments
Uploaded: 2010-09-05T17:00:00
Duration: 6 min 13 s
Description: Watch this Scientific Journal Video about Electroeluting DNA Fragments at JoVE.com

<p class="jove_content">Work in Dr. Bermudez laboratory was funded by Conacyt (Grant # 82622) .We thank Gabriela Gutierrez-Sosa, Eliuth Reyes-Martinez and Ximena Rodriguez-Torres for video recording the electroleution procedure.</p>

<ol>
	<li>In order to select DNA fragments of interest to be purified, these should be resolved in agarose or acrylamide gels gel stained with ethidium bromide by electrophoresis using 0.5X TBE buffer (Tris borate, EDTA) at 120 volts for 1 hour. For this, ~ 700 ng of plasmid pProEx-GdMre11S (6000-bp) previously digested with NdeI and HindIII was used to release a 900-bp fragment (560 ng of plasmid and 84 ng of fragment).</li>
	<li>The electroeluter tank (Figure 1) should be filled with 0.5X TBE equally distributed in each side of the mid plateau taking care of not spill it onto the mid plateau.</li>
	<li>The selected band (or bands) is cut out of the gel, and placed in sample chamber as close as possible to the V channel (one slice per well). Running buffer should be added to each chamber; just enough to cover the gel slice.</li>
	<li>Each V channel used must be flushed with a Pasteur pipette to eliminate any air bubble trapped.</li>
	<li>Then 100 ul of 10 M NH<sub>4</sub> acetate (to facilitate visualization a small amount of bromophenol blue is added, just enough to color it) are gently added inside the V channel.</li>
	<li>The lid should be closed gently to prevent any resuspension of the high salt cushion.</li>
	<li>Electroelution is begun at 100 volts for 25 minutes.</li>
	<li>When electrolution is completed, 400 ul are removed from V-channel, and placed in a microfuge tube to be precipitated with 2 volumes of cold ethanol and glycogen (recommended to improve DNA recovery) at 4 &deg; C for 1 hour or overnight.</li>
	<li>Centrifuge for 20 minutes, remove supernatant and wash with 70% ethanol.</li>
	<li>Dry tube and quantify DNA at OD 260 nm.</li>
	<li>The recovery obtained was 75% (63 ng recovered when 84 ng were placed on the V-channel).</li>
</ol>
<p class="jove_content"><img src="/files/ftp_upload/2136/2136fig1.jpg" alt="Figure 1" /><br /><strong>Figure 1.</strong> Electroeluter diagram. Anod an cathode are indicated on each side of tank, DNA contained in gel sliced  (illustrated as a black square) will migrate towards cathode due to its negative charge. Then it will be trapped in the salt cushion (represented as an inverted black triangle) located in the V-channel.</p>

<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> <em style='font-style: italic'>Molecular Cloning: A Laboratory Manual</em>. , (1989).</li></ol>

<p class="jove_content">No conflicts of interest declared.</p>

<p class="jove_content">The duration of the run will depend highly on the size of the fragments, normally for fragments up to 20 kb 50 minutes to 1 hour is enough, while for small fragments UV light monitoring every 10 minutes is required to prevent the fragment to go through the salt cushion and consequently to the anodal buffer chamber decreasing recovery. It is important to make sure that when you set your power supply at 100 volts, there is a current at least of 10 mAmp. The DNA band can be monitored by using a UV-hand lamp and detect when this abandons the gel slice. The cushion salt can also be made with 3 M Na acetate.</p>
<p class="jove_content">This procedure allows the purification of DNA or RNA fragments of different sizes with good recovery (~80%) to be radiolabeled, digested by restriction enzymes, processed by modifying enzymes, etc.</p>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NdeI</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>R0111L</td>
<td>&nbsp;</td>
<tr>
<td>HindIII</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>R0104L</td>
<td>&nbsp;</td>
<tr>
<td>NH<sub>4</sub> acetate</td>
<td>&nbsp;</td>
<td>JTBaker</td>
<td>0596</td>
<td>&nbsp;</td>
<tr>
<td>agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>
<tr>
<td>Biophotometer</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>6131 000 020</td>
<td>&nbsp;</td>
<tr>
<td>Electroeluter</td>
<td>&nbsp;</td>
<td>IBI</td>
<td>UEA CAT 46000</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

electroeluting dna fragments

<p class="jove_content">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.</p>

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

Electroeluting DNA Fragments

<p class="jove_content">This procedure allows the purification of DNA fragments with high yield.</p>

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

Research

JoVE Journal

Biology

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

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 ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

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 ...

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 ...