Gel electrophoresis

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I got more confused...  Is there possibly a more simple, maybe high school level explanatory? Or is that one already as simple as it gets, and I'm just being a noob? 

Gel electrophoresis is used to separate out proteins or DNA by size. Both proteins and DNA have an overall negative charge, so an electric field is used to pull the proteins/DNA towards the positive electric terminal. The gel matrix they are being pulled through is somewhat thick, so small pieces will travel further than bigger pieces. The steps vary according to what type of electrophoresis you are doing (for example, you can use proteins in their natural state or you can denature them so they are all unfolded), but this is the basic procedure:

1. Assemble the electrophoresis apparatus. This consists of two glass plates, spacers in between the glass plates, and some type of box with a positive and negative terminal.

2. Pour the gel in a liquid form (can use polyacrylamide or agarose) in between the glass plates. Insert a well-comb in the top between the plates, at the negative end.

3. Wait for the gel to solidify.

4. Pull out the comb such that wells for your solutions are left behind.

5. "Load" your samples of DNA or protein into the wells of the gel (basically dips in the gel that hold the sample solutions)

6. Fill the apparatus with an electrolyte buffer.

7. Turn on the apparatus. This will create an electric field that will pull your protein/DNA down through the gel matrix towards the positive end.

8. Usually a tracking dye is added to the sample solutions. The dye molecule is very small, so it will travel ahead of your proteins/DNA. When it reaches the end of the gel, it is time to turn off the apparatus.

9. Separate the glass plates and carefully remove the gel.

10. Dyes can be added to view the "bands" of protein/DNA. Each band is a protein/DNA type of a different molecular weight, with the smallest proteins/DNA traveling farthest through the gel.

11. You can also add a molecular weight standard to one of your wells before running the gel. This is a mixture of proteins or DNA of known molecular weights. By measuring how far they travel (ie where the bands show up), and comparing to your unknown protein/DNA sample bands, you can calculate the molecular weight of the protein/DNA in your sample.

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Gel electrophoresis is a technique used for the separation of biological molecules.. now basically electrophoresis refers to moving of charged particle in an electrical field..

thus it enables to sorting of molecules by size and charge..different types of gels are used for separating different molecules..

After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or Brilliant Blue dye may be used for this process.

In simple terms, gel electrophoresis works based on the polarity (electric charge) and size of the DNA fragments. Since DNA is negatively charged, it is placed on the negatively charged side of the plate. Since opposites attract, the negatively charged DNA would want to move to positive side of the plate ASAP. The smaller fragments are able to travel to the positive end much faster than the larger ones, hence, separating the DNA based on the size.

DNA is a negatively charged molecule. That means that as a compound, it has excess electrons (which are negatively charged). Basic physics says that like repels like, and opposites attract. So, gel electrophoresis (GE) exposes DNA in solution to an electric charge. This causes it to move.

GEs have an electrical set up so that the positive charge is running through a wire at the bottom of the set up and negative is at the top. So you make the gel, with little holes (wells in it) running parallel to the top and bottom where the charges will be. You cover the now solid gel with running buffer ( A salt solution that lets things run smoothly). Load your sample with a loading buffer ( to make the DNA sink into the well) and a ladder.

( DNA ladders are already made samples bought from companies. They consist of DNA of known sizes to allow you to calculate the size of the fragments in your sample). When you examine the gel after it has run enough, all bands along the same horizontal line should be the same size, as the movement through the gel is directly proportional to the size of the fragment. Bigger DNA pieces move slower, so will closer to the top as they didnt get very far from the well where they started.

Then you turn the voltage on. Typically, you would run a 2 % TBE / TAE gel for about 30 - 35 minuted at 100 V. You check its progress by examining the gel in a visualizer that uses UV light. A compound called ethidium bromide was also added into the gel when it was made and the running buffer too sometimes. This binds to DNA and makes it glow under UV.

1. There is a permeable gel with a row of holes on one side.
2. Different DNA samples are placed in holes using pipette.
3. An electric current is sent through the gel, with the opposite side as positive (the DNA is negatively charged).
4. Since the larger parts of DNA cannot get far through the gel and the smaller parts can. Several bands will be formed on the gel.
5. The bands are visible under UV light
6. These bands can be used to compare genetic similarity to determine the parent/sibling.

4.4.2 State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size.(1)

State means to give a specific name, value or other brief answer without explanation or calculation.

• Sample of fragmented DNA is placed in one of the wells on the gel.

• An electrical current is passed across the gel.

• Fragment separation is based on charge and size.

• Large fragments move slowly.

• Negative fragments are moved to the right.

 

 

Gel after staining:

• This diagram shows the separation of 6 separate mixtures of DNA.

• The dark bands to the left are those with a large molecular mass or a positive charge

• (a) contains 5 fragments of DNA. Each bands corresponds to a group of DNA molecules of the same size and charge.

• (b) and (c) have the same bands. They are identical