Name: Electroeluting DNA Fragments (Video) | JoVE Journal
Uploaded: 2010-09-05T17:00:00.000+00:00
Duration: 6 min 13 s
Description: Learn electroelution for DNA fragment purification using gel electrophoresis and alcohol precipitation. This method achieves up to 80% efficiency for cloning and sequencing applications.

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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.

<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 NH4 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 ° 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>
<img src="/files/ftp_upload/2136/2136fig1.jpg" alt="Electrophoresis diagram, gel slice in V-channel, anode to cathode setup, DNA separation process." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. 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.

<ol><li>Sambrook, J., Fritsch, E. F., Maniatis, T. Molecular Cloning: A Laboratory Manual. , 2nd Ed, Cold Spring Harbor Laboratory Press. 3 Volume Set (1989).</li>
</ol>

No conflicts of interest declared.

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.
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.

<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

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 ...

Hi.Today we'll Show you how electro lution is performed in order to purify DNA fragments such as digestion and PCR products. For further purposes like cloning and sequencing, electro lution can be one of the methods of choice. Based on the negative electric charge of DNA, A high cell barrier is placed within the electro field, dropping the fragments and preventing their further migration.This method allows the purification of a wide range of DNA fragment sizes from different sources with a relatively high efficiency of up to 80%Its low cost. Represent another advantage of electro lution when compared to other purification methods such as columns, resin kits, among others. The DNA sample is first visualized by a tedium bromide stained Aris gel electrophoresis in order to visualize and identified the desired fragment to be purified, electrophoresis should proceed.Under fragments are well separated, undersized can be precisely determined. Following electrophoresis. Desired fragment is neatly excised from the gel using a brand new razor blade and taking care to turn the gel slice as close as possible to the band of interest.This procedure should be performed With proper protection from UV light and wearing gloves. After filling the electro luter with the Appropriate burning buffer and taking care to spill it over the surface of the middle plateau, the gel slice is placed inside one of the wheels as close as possible to the V channel. There should be no bubbles inside the vch channel.This can be avoided by flushing the channel with buffer to remove any trapped air. Then 100 microliters of the high salt barrier are added carefully to the V channel from the site proximal To the well. The chamber is Enclosed and the electrodes connected to the power source to begin electro lution at 100 to 120 volts.The time lapse will depend on the fragment size for large fragments of up to 20 kbs. 50 minutes to one hour should be enough and small fragments should monitor every 10 minutes with UV light to avoid further elion through the salt solution and into the end adult buffer chain. In this case, we digested 560 nanograms of plasmid with the enzymes ND one and entry to release a 900 base per fragment, a equivalent to 84 nanograms of a sample.Accordingly, after placing the gel slice inside the well, electro lution will proceed for 25 minutes at 100 volts. Once electro solution is completed and DNA fragments are trapped in the south cushion within the V channel, 400 microliters of solution are recovered from the channel, from the distal side of the receptacle And Placed in a 1.5 microliter micro tube using a fine tip attached to a micro pit, proceed to alcohol precipitation by adding two volumes of cold absolute ethanol and mixed by vortex. Additionally, two to three microliters of glycogen may be added to improve the yield Of DNA recovery classic tubes At minus 20 degrees Celsius for one hour or alternatively, store at this temperature overnight centrifuge at four degrees Celsius at top speed for 25 minutes.This cardio supernatant and wash by adding one milliliter of cold, 70%ethanol Centrifuge at four degrees Celsius at top speed for five to 10 minutes, discard supernatant and allow to dry it room temperature with the tubes in an inverted position, greases spent in 10 to 15 microliters of nucleus free water and proceed to determine the concentration of the sample. In this case, the purified sample was resuspended in 10 microliters of water and quantified in a spectrophotometer at 260 nanometers wavelength showing a concentration of 6.3 nanograms per microliter, representing a 75%efficiency. You may be thinking you'd be better off investing only 10 minutes of your precious time with a kit.However, in spite of the time consumed in this process, you will be able to control many of the variables that will allow you to purify a wider range of samples bearing in nature sizes and sources. As you could have seen in the present case, a 75%efficiency was observed equal to or better than most DNA purifying kits. Thank you for joining us today.We hope that you have found this protocol useful for your research Projects.

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Learn electroelution for DNA fragment purification using gel electrophoresis and alcohol precipitation. This method achieves up to 80% efficiency for cloning and sequencing applications.

Electroeluting DNA Fragments

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