实验概要

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.

实验步骤

Dissolving Agarose 1000 (<3%)

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.

Dissolving Agarose 1000 (4%-5%)

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.

Dissolving Agarose 1000 (>5%)

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.