Good job keeping to an encyclopedic style that is accessible. Also, JoVE is a great source, since videos help for methods, but try to find secondary literature or other protocol papers.BenjaminLaufer (talk) 18:25, 3 November 2015 (UTC)
This is a user sandbox of Kathryn.cc. A user sandbox is a subpage of the user's user page. It serves as a testing spot and page development space for the user and is not an encyclopedia article.
Article Topic -> electroelution
Sources ->
explains protocol via written process, diagram and video. used to increase understanding of the process both for the article page as well as my understanding so I can write on a topic that i'm educated on [1]
relatively recent paper that compares electroelution to other DNA/protein extraction methods. used to show that this process can be used to extract multiple different molecules from gels and explain the benefits over other processes [2]
not too sure if i should use to explain benefits over other processes because I think this isn't in keeping with the encyclopedia format
describes application of electroelution in BAC (bacterial artificial chromosome) and PAC (P1-derived artificial chromosome) cloning and shows utility of electroelution in terms of DNA purification (essentially what BAC and PAC cloning are) [3]
comparison was made in existing article and felt i should keep it
Overview of Current Stub -> The current stub does a good job of explaining the process of electroelution (cutting gel and using electricity), but it is unclear exactly how the process works and it gets a bit confusing and difficult to picture the process.
Overview of Future Article -> Things I intend to add/change:
-add a diagram of the process
-explain the process step by step (possibly with a diagram at each step)
-add important uses (what it can be used to extract)
The major change that I will make to the article is to have it written at a level that people without a science background can understand it. To do this, I will include a step-by-step process of how electroelution is done. I will also add in visual aids of the machine itself and the process (whether it be one diagram showing the overall process or one image per step). I will also ensure to add a list of electroelution's utilities (for example, what types of molecules it is able to extract/purify).
Possible Headings ->
-Step-by-Step Extraction Process (w diagram(s))
-Utility
Sentences for Future Article ->
for the "Utility" heading: Electroelution is able to extract multiple molecules types such as; DNA, RNA, proteins, RNA-protein complexes and DNA-protein complexes.
for the Step-by-Step Extraction Process heading: The molecule of interest is removed from the agarose/polyacrylamide gel using a sharp edge, taking care to cut as close to the band of the molecule as possible.
Electroelution is a method used to extract a nucleic acid or a protein sample from an electrophoresis gel. It is done by applying a negative current through the gel.[5] This draws the macromolecules (nucleic acid or protein) to the surface of the gel. The molecule is then drawn out of the gel and caught in the high-salt barrier solution inside the collection chamber/V-channel of the electroelution device (shown below). [5] The molecules can then be extracted and analyzed. The goal of this process is similar to that of electroblotting.[6]
Visualize the sample on gel using a stain and identify the band of molecular fragments to be purified
Excise the section of the gel containing the molecule of interest with a sharp razor blade
Cut as close to the band as possible, reducing excess gel
Place the excised gel fragment inside a small tube (such as an Eppendorf tube)
This step allows for only the molecule of interest to be isolated.
Preparing for Electroelution
Electroelution chamber with the gel fragment, high salt barrier and buffer in placeFill electroeluter with appropriate buffer solution so that the liquid buffer is level with the channel of wells in the middle of chamber (as shown in the diagram in pale green)
Place the gel fragment inside one of the wells
Add a small amount of a liquid high salt barrier solution to the V-channel **(so that the electroeluter chamber looks like the one shown)
This step prepares for the electroelution process, placing each component in its respective location.
Running Electroelution
Running electroelution (dotted lines and arrows indicate flow of electricity)
Close chamber, attach electrodes and turn on power source (100-120V)
Allow the current to flow for a time relative to the size of the molecule of interest (smaller molecule, less time; larger molecule, more time)
Approximately 30 minutes to an hour should suffice
**DNA/molecule of interest is caught in the salt barrier solution within the V-channel
This step is what extracts the molecule of interest from the gel.
Extracting the Molecule of Interest
Turn off power, open the chamber and remove salt barrier from V-channel
Place in a small tubeExtraction of high-salt barrier
Separate out the molecule of interest from the salt barrier solution by precipitating it out
This is done by adding ethanol and mixing by vortexing
A solid pellet should remain at the bottom of the tube
This step allows the molecule of interest to be transferred and used for other experiments.
VOILA! You have isolated DNA/molecule of interest
Utility/Potential Uses
Electroelution can be used for a variety of purposes. Three main applications include: extraction of macromolecules, reduction in cost of cloning, and serving as an alternative method to isolate macromolecules.
Electroelution is able to extract a variety of macromolecules following gel electrophoresis.[1][2][4][5] Examples of these molecules are: protein, DNA, RNA, protein-RNA complexes, and oligos (for PCR, cloning and primer extension).
Using a bacterial based system (BAC and PAC), electroelution allows for the cloning of large DNA fragments to be done at a reduced cost. [3] This is because it allows DNA isolation without the need for heat. Electroelution also produces higher molecule yields (has a higher efficiency).
Electroelution is an alternate for many other methods that also isolate macromolecules, such as protein or DNA. These methods that can be replaced include: dissolving the gel using heat and other methods that may destroy the macromolecule of interest[3][5], using a gel that is capable of dissolving in acid[5], “crush and soak” method where the gel is blended into a slurry-like consistency and then soaked[5], and expensive commercial methods using complicated machinery[5].
Benefits
Electroelution has many benefits. It has a simple and easy construction design.[5] The electroelution process is low cost as it doesn’t use any expensive or overly fancy equipment and thus keeps costs down.[4][5] Also, it has a relatively high efficacy. The recovery rate for proteins is 60% or more[1][4] while it is more than 90% for DNA.[4] The process is quick. If need be, electroelution can be done in as little time as 20 minutes ~2 hours if need be.[1][4] Finally, electroelution allows for the extraction of short sequences. It is capable of removing even small fragments of molecules from gels. For example, a 15 nucleotide long sequence can be extracted from the gel.[4]
