High Resolution Agarose Gel Electrophoresis

Introduction

Agarose gel electrophoresis remains the most widely used technique for separating nucleic acid fragments due to its ease of use, non-toxicity, and broad separation range. By varying agarose concentration, gel pore size can be controlled to separate nucleic acid molecules in a wide range of sizes. The migration of nucleic acids in agarose gels is affected by the choice of buffer and applied voltage. Invitrogen offers a range of UltraPure™ agarose products to meet your nucleic acid electrophoresis needs. As with all Invitrogen UltraPure™ reagents, these products are made from the highest purity biochemicals for maximum reliability and superior performance. In addition, every lot is tested to ensure that each UltraPure™ agarose product:

• Forms a homogenous, clear gel and separates the nucleic acid fragments into distinct bands
• Has very low electroendosmosis to minimize internal convection and band spreading during electrophoresis, allowing for sharp band resolution
• Has no detectable DNase or RNase activity

Agarose 1000 is a specialized agarose that exhibits the following properties:

• high resolution of PCR products and other short DNA fragments
• better handling because of a stronger gel structure
• improved clarity of the gel, enhancing visibility
• excellent mechanical strength 

NOTE:   Chill Agarose 1000 gels for 30 minutes at 4°C before use to achieve best resolution.
 
Method 1: Microwave (recommended for < 3% concentrations)

1. Into a flask holding 2-4 times the desired solution volume, add a magnetic stir bar and the calculated volume of buffer at room temperature.


2. Put the flask on a magnetic stirrer and slowly sprinkle the calculated amount of agarose powder into the flask while stirring constantly to prevent the formation of agarose clumps.


3. Remove the stir bar


4. Weigh the flask and solution before heating.


5. Place in the microwave oven and heat on high power for two minutes.


6. Remove carefully as any microwaved solution may become superheated and foam over when agitated. Gently swirl to resuspend any agarose particles.


7. Reheat on high power using 15-20 second intervals or until the solution comes to a boil, and solution is complete.


8. Remove carefully and gently swirl.


9. Return the flask to its original weight by adding warm distilled water.


10. Mix gently and cool to 50-60°C (at room temperature for at least 20 minutes) before pouring the solution into the tray.


11. During the cooling time any air bubbles will disappear.

Method 2: Boiling water bath (recommended for all concentrations, especially 4%-5%)

1. Into a flask holding 2-4 times the desired solution volume, add a magnetic stir bar and the calculated volume of buffer at room temperature.


2. Put the flask on a magnetic stirrer and slowly sprinkle the calculated amount of agarose powder while stirring constantly to prevent the formation of agarose clumps.


3. Weigh the flask and solution before heating.

• Bring the solution to a boil while stirring and allow to gently boil for approximately 15-20 minutes or until the agarose dissolves completely.
• Return the flask to its original weight by adding warm distilled water.
• Mix gently and cool to 50-60°C (at room temperature for at least 20 minutes) before pouring the solution into the tray.
• During the cooling time any air bubbles will disappear.

 Method 3: Autoclave (recommended for all concentrations, especially > 5%)

1. Into a flask holding 2-4 times the desired solution volume, add a magnetic stir bar and calculated amount of buffer at room temperature.


2. Put the flask on a magnetic stirrer and slowly sprinkle the calculated amount of agarose powder into the flask while stirring constantly to prevent the formation of agarose clumps.


3. Heat two minutes in the microwave oven at medium power.


4. Cover the opening of the flask with an aluminum foil to prevent spillover and autoclave at 121°C for 15 minutes.


5. Remove from the autoclave and allow to cool to 50-60°C before pouring the solution into the tray.