Agarose Gel Electrophoresis

实验概要

Separating nucleic acid fragments by agarose gel electrophoresis.

实验原理

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:

1.         Forms a homogenous, clear gel and separates the nucleic acid fragments into distinct bands

2.         Has  very low electroendosmosis to minimize internal convection and band  spreading during electrophoresis, allowing for sharp band resolution

3.         Has no detectable DNase or RNase activity

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

1.         high resolution of PCR products and other short DNA fragments

2.         better handling because of a stronger gel structure

3.         improved clarity of the gel, enhancing visibility

4.         excellent mechanical strength

实验步骤

1.        Dissolving Agarose 1000 (<3%)
 

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.

2.        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.

4)      Bring  the solution to a boil while stirring and allow to gently boil for  approximately 15-20 minutes or until the agarose dissolves completely.

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

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

7)      During the cooling time any air bubbles will disappear.

3.        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.

注意事项

Chill Agarose 1000 gels for 30 minutes at 4°C before use to achieve best resolution.