Making an Agarose Gel - University of Leicester

agarose gels are used in molecular biology for the separation and purification of nucleic acid fragments a dye is added so that the fragments can be visualized under ultraviolet light the samples are loaded into the wells of the gel and separated by size using an electric current the size range of fragments that can be separated using agarose is between about 9.2 kilobases and 20 kilobases agarose gels can be made with varying concentrations of agarose between Noor point six percent and three percent depending on the size of the fragments you wish to separate or resolve smaller fragments are resolved better in a gel with a higher percentage of agarose and larger fragments are resolved better in a gel with a lower percentage of agarose there are two basic components to an agarose gel the agarose which is a white powder and a buffer solution these need to be heated together to make the gel and preparing an agarose gel you'll need to both weigh out the appropriate amount of agarose and pour out the appropriate volume of buffer solution which will vary depending on the size and agarose concentration of the gel combine the agarose and buffer solution and mix them to make sure that no large lumps of agarose powder are stuck to the container the next step is to heat the agarose and buffer mixture in a microwave oven until the agarose is completely dissolved make sure the container holding the gel mixture is large enough to allow for the solution to boil up without coming out of the container a loose cover should be placed over the top to prevent the solution from splashing out it's very important that this cover be loose otherwise dangerous pressure buildup can occur within the container you should mix the gel solution at intervals during heating but be sure you wear safety gloves the solution can become superheated and when you mix it the liquid can suddenly boil up the agarose is only fully dissolved when it's completely clear if you don't completely dissolve the agarose you're finished gel will have regions of different concentrations and your nucleic acid samples will not separate correctly once the agarose is fully dissolved let it cool to 60 degrees Celsius this prevents heat damage to the gel tray while keeping the agarose liquid once the agarose is cooled you'll need to add a die to the molten gel for later visualization of the size separated nucleic acid fragments you can use cyber green but the best and most commonly used die is etherion bromide when using etherium bromide you should always wear gloves since it's a mutagen and a possible carcinogen that can be both absorbed through the skin and breathed in immediately after handling aetherium bromide change your gloves to avoid contaminating other instruments or parts of the lab gel trays have two open ends that need to be sealed while the gel is solidifying this is easily achieved using masking tape take a piece of masking tape that's a few centimetres longer than the open end of the gel tray and fold over one end of the tape to make it easier to remove later place the tape around the end of the gel tray overlapping both sides of the tray and make sure it's firmly stuck along the entire end do the same for the remaining open-end before pouring your gel you need to make sure the gel tray is on a horizontal surface so that gel will be of uniform thickness you also need to insert a comb into the gel tray the comb will create cavities called wells it's into these wells that you'll be preparing your nucleic acid samples once you've made sure that the gel tray is level and you've inserted the comb slowly pour the gel solution into the gel tray using a disposable pipette tip move any air bubbles to the edge of the gel it's especially important to remove bubbles from around the combs since these can affect the shape of the wells it takes about 20 minutes for the gel to solidify during which it will change from colorless to slightly opaque while the gel is setting don't move the gel tray or disturb the gel itself since this can create a gel of non uniform thickness once the gel is set you can remove the comb slowly and carefully pull the comb upwards vertically you'll feel some resistance as the comb comes out of the gel then remove the masking tape from the ends of the gel tray

Channel: University of Leicester

Views: 171,330

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Keywords: Agarose Gel, GENIE, University of Leicester, Genetics, Raymond Dagleish, Nicola Suter-Giorgini, Cas Kramer

Id: wXiiTW3pflM

Length: 5min 44sec (344 seconds)

Published: Fri Jun 26 2009