Agarose gels are used for gel electrophoresis to visualize DNA with the assistance of ultraviolet light. To make them, agarose is added to an electrolyte buffer ([[TAE|TAE]] or TBE) and the solution is boiled, melting and dispersing the agarose. A visualizing agent (e.g. [[Ethidium bromide|ethidium bromide]]) is added to the solution, which is then poured into a casting tray with combs in place. The solution cools until solidified, after which the combs are removed, leaving wells to which sample may be added. Gels are then ready for use. ## Procedure > [!danger]- Safety > Always wear appropriate PPE, including gloves and a lab coat. Handle hot glassware with heat-resistant gloves and add [[Ethidium bromide|ethidium bromide]] in a chemical fume hood. For more information on [[Ethidium bromide|ethidium bromide]], review its linked note. | Application | Gel length (cm) | [[TAE\|TAE]] (mL) | % agarose (w/v) | Agarose (g) | Width | | --------------------- | --------------- | ----------------- | --------------- | ----------- | -------- | | Routine visualization | 7 | 40 | 1 | 0.4 | standard | | Routine visualization | 7 | 100 | 1 | 1 | wide | | Routine visualization | 10 | 50 | 1 | 0.5 | standard | | Gel extraction | 7 | 40 | 0.8 | 0.32 | standard | | Gel extraction | 7 | 100 | 0.8 | 0.8 | wide | 1. Refer to the recipe table to choose an appropriately sized gel for your application. 2. Use a clean spatula to measure the agarose into a weigh boat.[^1] 3. Transfer the agarose into a flask and add the appropriate volume of buffer (usually TAE) into the flask.[^2] 4. Microwave the solution in 1-minute intervals at 50% power, using heat resistant gloves to swirl the flask between intervals until the agarose is fully dissolved; the solution will appear completely clear. Monitor the process closely, stopping the microwave to swirl and check the solution any time a rapid boil is sustained. 5. Take the flask to the chemical hood and add a 5 mg mL$^{-1}$ [[Ethidium bromide|ethidium bromide]] stock solution[^3] to a 0.5 µg mL$^{-1}$ concentration (e.g. a 0.1% (v/v) addition; add 4 µL to a 40 mL gel). 6. Prepare the casting tray and place the combs as desired. Pour the agarose into a casting tray and allow it to solidify (~30 minutes for 40 mL gels or 40 minutes for 100 mL gels).[^4] 7. Once the gel is solidified, remove the combs slowly but firmly to avoid breaking the gel. ## Storage [[Ethidium bromide|Ethidium bromide]] gels can be stored for at least a week at 4°C, provided they are not allowed to dry out and are kept out of light. To this end, premade gels can be stored in a dark refrigerator in an opaque Tupperware or similar container, with a thin layer (~1/4 inch) of the same buffer used to prepare the gel coating the bottom of the container.[^5] [^1]: If you add too much, remove it using the same spatula and discard the removed agarose into the trash — do not return it to the bottle. [^2]: In this procedure, the volume contributed by the agarose is negligible and may be ignored. Even if you wanted to account for this volume, volumetric flasks rarely have enough graduations to make such an accurate measurement, making it impractical to account for the volume of the powder as one would normally do to prepare a solution. [^3]: [[Ethidium bromide|Ethidium bromide]] is solubilized and stored in the refrigerator in a light-proof tube, usually a 1.5 or 5 mL microcentrifuge tube wrapped in aluminum foil. In our laboratory, this can be found in the door of the GE refrigerator in BSBE 320. To prepare the stock solution, see the [[Ethidium bromide|ethidium bromide]] page. [^4]: Immediately after pouring, while the residue in the flask is still molten, take the flask it to the sink and rinse it to remove as much agar as possible. [^5]: A stored gel should not be submerged, nor be close to submersion, as the[[Ethidium bromide|ethidium bromide]] in the gel will leach into the buffer during storage. As long as the buffer in the container is a sufficiently small volume, the effect is negligible on visualization, but larger volumes of storage buffer can substantially dilute the [[Ethidium bromide|ethidium bromide]] that you need to stay in your gel. To this end we have found 1/4 inch layer of buffer in the container is sufficient.