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Gel Electrophoresis in Molecular BiologyBY: Lorato Lekgari | Category: Biology | Submitted: 2010-07-25 14:44:38
Article Summary: "Discusses gel electrophoresis and outline the protocol that can be used. A gel, which is a matrix with a network of pores that allows molecules to travel, is thus used as a way to contain and then separate molecules based on their size..."
This is a separation technique that separate components of molecules based on size and charge. This is possible because most bio molecules have an inherent charge thus they will migrate towards pole of opposite charge in an electric field. It is used as an analytical or preparative tool. Most molecules have a similar charge and mass ratio and thus cannot be separated by standard in liquid solution electrophoresis. A gel, which is a matrix with a network of pores that allows molecules to travel, is thus used as a way to contain and then separate molecules based on their size. The smaller molecules will therefore move faster thus further down the gel as compared to large ones. There are different gels used to separate different kinds of molecules.
Different Gels Used
Agarose gel electrophoresis is used to separate large DNA molecules of the size range1-30kilobases. This is because it has large pores. The separation can also depend on the concentration of the agarose in solution. The gels are mostly 5mm thick and 0.5% and 1% are used. For small DNA molecules of sizes ranging from 1 to 300 base pairs can be separated using 40% polyacralamide gel. These gels are 0.3mm thick and are able to distinguish between DNA fragments that ere 1bp different in length. RNA as is composed by nucleic acids which carries a negative charge just like DNA, is thus separated in the same way as for DNA. Proteins on the other hand, can have complex shapes and varying charges thus making it difficult to separate them by just using standard gel electrophoresis. They are thus treated first by being denatured and coated by detergents such as sodium dodecyl sulphate (SDS), to give then a negative charge. Thus proteins are analysed in using sodium dodecyl sulphate polyacralamide gel electrophoresis (SDS-PAGE).
Visualising the separated molecules
The electrophoresed molecules have to be visualised on the gel so that their sizes can be determined. This is done mainly by staining the gel or by autoradiography. Staining is the simplest method and ethidium bromide is mainly used for staining. Ethidium bromide intercalates into the nucleotides and it fluorescence under ultra violet light thus allowing for the DNA/RNA bands to be visualised. Proteins are visualised by being stained with Coomassie brilliant blue which binds non-specifically with the protein molecules will appear as blue bands on a transparent bare ground. Autoradiography can be used to visualise DNA/RNA and protein molecules. This is done by radioactively labelling the molecules before electrophoresis and then an X-ray film can be put over the gel to visualise the bands.
Tracing Electrophoresis and Determining Sizes of the Separated Molecules
One must be able to trace the movement of DNA/RNA and protein molecules as they are electrophoresed. To be able to do this a loading buffer is used. Loading buffers mainly serves the purpose of giving the molecules colour and density. The dyes mainly used are bromophenol blue and xylene cyanol where as glycerol or sucrose can be used to provide the density that will make the sample to sink into the gel wells. The dyes are negatively charged and thus move on the same direction as the molecules being separated and they show how far gel electrophoresis has gone. A molecular weight marker is used to be able to determine the sizes of the different DNA bands after separation. The different sizes of the molecular weight marker have to be known thus there are standard molecular weight markers such as lambda PstI that can be used.
DNA Gel Electrophoresis Standard Protocol
To prepare 60ml of a 1% agarose gel;
1. Weigh 0.6g of agarose and add 60ml of 0.5x TBE (Tris Borate EDTA) buffer. Dissolve the agarose in the microwave until granule cannot be seen in solution.
2. Allow the solution to cool to about 60°C.
3. Whilst waiting for it to cool down, prepare the gel tray making sure it is clean and place a comb that is going to make wells. Depending on the number and quantity to be loaded, an appropriate comb can be chosen e.g. For a 60 ml gel to load 25µl of sample, a comb with 8 big teeth can be used.
4. Then add 1.2µl of ethidium bromide to the cooed solution swirl to mix then pour into the prepared gel tray.
5. Leave the solution for 10-20 minutes to solidify
6. Prepare the gel tank by pouring 0.5x TBE buffer into it to a volume that the gel will be able to be submerged in it to a depth of about 5mm
7. Prepare the DNA sample by adding loading buffer to it i.e. add 4µl loading buffer into 20µl sample.
8. Then load the samples into the gel wells but first load the molecular weight marker followed by the samples using a micro pipette.
9. Put the gel into the gel tank close it. Make sure that the electrodes have been appropriately connected, remember DNA is negatively charged and it will move to the positive charge. Then switch on the power.
10. Monitor the movement of the loading buffer to keep track of the DNA movement
11. Then after electrophoresis, take the gel out and visualize under UV light in a UV cabinet. A photograph of the image can be taken if a camera is connected to the UV cabinet.
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