Agarose I

EMS10205, EMS10207

TIPS FOR PREPARING 

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 polymerised.

Guide 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 polymerise 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)

SEPARATION OF BIOLOGICAL MOLECULES BY GEL ELECTROPHORESIS

Protocol Overview

1. In a flask 2-4 times greater than gel volume add agarose to buffer with constant swirling.
2. Weigh flask
3. Microwave on high for 30 seconds
4. Swirl solution
5. Microwave on high for 30 seconds
6. Swirl solution
7. Microwave on high until solution boils
8. Remove and allow to cool for 1-2 minutes before swirling solution again
9. Boil again for 15 seconds
10. Check for crystals and repeat boiling until solution is homogenous
11. Weigh flask and add water to return to original weight
12. Cool to 50-55°C
13. Swirl and pour into casting stand
14. Allow 30-45 minutes for gel to solidify

Microwave instructions:

Recommended for agarose concentrations ≤ 3%.

1. Agarose must be uniformly dispersed in buffer prior to hydration to avoid clumping.
   
   Agarose I tablets should be crushed into a powder with a clean rod or spatula prior to dispersion. 
   
   Determine gel volume and agarose concentration:
   
   Estimate volume:
   Volume = surface area of the casting chamber X gel depth
   
    Optimal resolution is usually obtained on gels 3-4 mm thick.
2. In an appropriate container (an Erlenmeyer flask at 2-4 times the volume of the desired gel volume) slowly add agarose crystals to your buffer solution while gently swirling the flask. 
3. Weigh the flask containing the buffer and agarose.                           
4. 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 the wattage of your microwave oven, size of the flask used and the  agarose concentration.
5. Swirl the agarose solution gently to re-suspend the particles.
6. Heat the solution another 30 seconds on high power, remove and swirl the agarose solution.
7. 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).
    Caution: Handle the hot flask very carefully.  Microwaved solutions may become superheated and boil over when moved or touched.  
8. Remove the boiling solution from the microwave oven, allow to cool briefly (1-2 minutes) at room temperature. Gently swirl the solution to release entrapped air (some air bubbles will remain).
9. 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 and the solution is transparent.
10. Once the agarose is completely in solution, reweigh the flask to check for water loss by evaporation. Replenish with hot distilled, deionised water until the weight of the flask and its contents equal the original weight. Gently swirl the solution.
11. Allow the agarose solution to cool at room temperature to ~50-55°C before pouring the gel into the prepared casting stand. This will result in a gel with a more uniform pore size and prevent warping of the gel apparatus. 

Notes: Preparation of any agarose solution in the microwave requires constant attention to prevent the solution from boiling over. 

• Gently swirl agarose solution at least twice before bringing the solution to a boil.
• Once the boiling point has been reached, observe the solution after each 10-15 second boiling interval until particles are no longer visible.

Hotplate/magnetic stirrer instructions:

Particularly recommended for agarose concentrations between 4%-5%. It may be used for lower concentrations as well.

1. Follow Step 1 for microwave instructions (above).
2. With heat off, place an appropriate container (an Erlenmeyer flask at 2-4 times the volume of the desired gel volume) containing buffer and a PTFE coated stir bar on the magnetic stirrer. With rapid stirring to prevent formation of clumps slowly add agarose to your buffer solution. 
3. Weigh the flask containing the buffer, agarose and stir bar.
4. With rapid stirring bring solution to a boil.
5. Maintain a gentle boil until agarose is completely dissolved, about 3-10 minutes. Inspect solution for agarose crystals and continue boiling if necessary. 
6. Follow steps 9-13 in microwave instructions above (above).

Autoclave Instructions:

Particularly recommended for agarose concentrations between >5%. It may be used for lower concentrations as well.

Caution: Agarose solutions containing Ethidium Bromide or other mutagenic intercalating stains should not be autoclaved to avoid apparatus contamination.  

1. Follow steps 1-6 for microwave instructions (above).
2. Cover flask with aluminium foil and autoclave at 121°C for 15 minutes.
3. Remove from autoclave and allow to cool briefly. 
4. Weigh the flask and add warm distilled water to return to original weight.
5. Allow to cool to 50-55°C before pouring into casting stand.

Gel Casting

1. Assemble casting stand according to the manufacturer’s instructions.
   • Casting stand should be level.
   • Comb teeth should be examined for dried agarose and cleaned with hot water and rinsed in distilled, deionised water prior to use.
2. When agarose solution cools to ~55°C, gently swirl to help dissipate most of the remaining air bubbles.
3. Pour the gel into the prepared casting unit to a depth of 3-4mm.  Immediately after pouring,  insert comb and check to see that there are no air bubbles under or between the teeth of the gel comb.
4. Allow the gel to completely solidify at room temperature (about 30-45 minutes).

Note: To achieve optimal resolution with Agarose SFR™: Allow SFR™ agarose gels to solidify completely at room temperature. The gel should then be placed at 4°C for 30 minutes prior to loading and running.