EMS #10205; 10207
When agarose is placed in a buffer such as TAE (Tris/Acetate/EDTA) or TBE (Tris/Borate/EDTA), it is generally insoluble. However, when this agarose solution is heated, the agarose particles become hydrated and thus go into solution. This hydration process is time-dependent, and different types of agarose will have varying hydration points. EMS’s Agaroses are extremely pure and comprised of ultra-fine particles. This ultra-fine structure, EMS agaroses will have a faster rate of hydration than other type of agaroses. End-users who have used other brands of agarose in the past may mistakenly boil EMS agaroses much longer than is needed, which results in a thick gelatinous solution that is difficult to cast and brittle when polymerized.
Helpful Hints for Preparing EMS Agaroses
Preparation of a typical 1% agarose gel 1X TBE buffer.
- In an appropriate container (an Erlenmeyer flask at 2 –4X the volume of the desired gel volume is optimal), slowly add agarose crystals to your buffer solution while gently swirling. This will help to eliminate clumping of the agarose. Record the weight of the flask containing the buffer and agarose.
- Heat the solution in a microwave on high power for 30 seconds (for smaller or larger volumes, increase or decrease heating times proportionally to volume size). Heating times will vary depending on your microwave oven (wattage), size of the flask used and the % agarose.
- Swirl the agarose solution gently to re-suspend the particles.
- Heat the solution another 30 seconds on high power, remove and swirl the agarose solution.
- Place the solution back in the microwave and heat on high power until the solution just starts to boil (boiling point will probably take 10-35 seconds). Use caution when handling the hot flask. Microwave solutions may become superheated and can be boil vigorously when moved or touched. After removing the boiling solution from the microwave oven, allow to cool briefly (1-2 minutes) at room temperature, then gently swirls the solution to release entrapped air (some air bubbles will remain).
- Place the agarose solution back in the microwave, heat on high power and let the solution boil for approximately 15 seconds. Inspect the solution for agarose crystals (they will appear as floating ‘lenses’) while gently swirling. If there are particles present, repeat this step until all crystals are dissolved.
- Once the agarose is completely in solution, again weight the flask to check for water loss by evaporation. Replenish with water as necessary (until the weight of the flask and its contents equal the original weight). Gently swirls the solution.
- In general, it is advisable to allow any agarose solution to cool to ~ 50 – 55°C on the lab bench prior to pouring into a prepared apparatus. This is conductive to a more uniform pore size and will prevent the warping of your gel apparatus. Before pouring the gel, gently swirls the agarose solution to help dissipate most of the remaining air bubbles.
- Pour the gel into the prepared casting unit. Usually, horizontal gels should be 3 – 5mm thick. Immediately after pouring, check to see that there are no air bubbles under or between the teeth of the gel comb.
- Allow the gel to completely polymerize at room temperature (about 30-45 minutes) before running your samples. For further information on agarose gel electrophoresis, see Sambrook et al. (Chapter 6).
If you are preparing a different type of agarose (i.e., a higher concentration or a different volume), the most important things to remember are:
- Gently swirls your agarose solution at least twice before you bring the solution to a boil.
- Once the boiling point has been reached, observe your solution after each 10-15 seconds, boiling intervals very closely (depending on concentration and volume)