showcase.par.72124.image.0.0.1

Agarose gel electrophoresis is a basic and essential technique in molecular biology. It is routinely used for analysis of PCR products, plasmid DNA, and products of restriction enzyme digestion. It is the first step for analysis of specific DNA and RNA fragments by northern and Southern blots. To perform agarose gel electrophoresis of PCR products, we have included two protocols: Using E-Gel EX agarose gels, and Using UltraPure Agarose.

Using E-Gel EX Agarose Gels

It couldn’t be easier to run this high-resolution agarose gel. All you need is 15 minutes, an E-Gel EX gel, and an iBase Power System.

Total Time:

15 min
(Prep: 5 min)
(Electrophoresis: 10 min)

Level of Difficulty:

Easy

Tools needed:

Pipette and tips

Cost:

~$14.00 USD (per gel run)


Ingredients


Directions

  1. Connect the iBase power cord to an electrical outlet. Open the E-Gel EX agarose gel package. Remove the comb.
  2. Slide the cassette into the electrode connection of the iBase power system. A steady red light will illuminate to show that the cassette is correctly inserted.
  3. Add 0.2 µL volume of loading buffer to the samples; e.g., 4 µL into 20 µL sample. Load 100-250 ng of your sample and add E-Gel loading buffer to 20 µL.
  4. Load the first and last wells with 20 µL of diluted markers/ladders (Use 5 µL of E-Gel marker plus 15 µL of water). Continue loading 20 µL of sample per well. (All wells in the gel must be loaded with either sample in loading buffer or water.
  5. Toggle between programs until program 7 (E-Gel EX) is reached. The default run time is 10 min. Press the Go button to start electrophoresis. A green light will illuminate to show the run is in progress.
  6. After the run, remove E-Gel EX cassette and visualize PCR fragment bands using the E-Gel Imager or other blue light source (Safe Imager 2.0 or E-Gel Safe Imager).

Using UltraPure Agarose

Discover classic agarose gel electrophoresis with UltraPure Agarose and reagents.

Total Time:

90 min
(Prep: 50 min)
(Electrophoresis: 40 min)

Level of Difficulty:

Moderate

Tools needed:

Balance; 1 L graduate cylinder; microwave; pipette and tips; water bath; power supply

Cost:

~$6.75 USD (per gel run)

 
Ingredients


Directions

  1. Weigh out 1 g of agarose gel and add it to a 500 mL flask. Add 100 mL of 1X TBE. (The total gel volume will vary depending on the size of the casting tray.)
  2. Gently mix and heat the solution in a microwave until the agarose is completely dissolved. (CAUTION: wear protective gloves when handling extremely hot agarose solution and heat the solution in several short intervals—do not let the solution boil for long periods as it may boil out of the flask or cause a loss to water vapor. You can weigh the flask before and after heating and add distilled water to make up lost volume.)
  3. Allow hot agarose to cool in a water bath set at 50–55°C for 10 min. Swirl the flask occasionally to cool evenly.
  4. Prepare the gel casting tray by sealing the ends of the gel chamber with tape. Place the 10-well comb in the gel tray. (Note: pour the gel on a level surface; otherwise, the gel thickness will not be even, resulting in uneven migration)
  5. Add 5 µL of stock ethidium bromide solution to the cooled gel and pour into the gel tray. Remove any bubbles and allow gel to cool for 30 min at room temperature. (Exercise caution when using ethidium bromide, which is a known carcinogen.)
  6. Remove the comb and tape, place the gel tray in the electrophoresis chamber, and cover with 1X TBE buffer. (Cover the gel with ~2–3 mm of buffer for best results.)
  7. Add 0.2 µL volume of loading buffer to samples; e.g., 4 µL of 20 µL sample. Load 100-250 ng of your sample and add E-Gel loading buffer to 20 µL.
  8. Load the first and last wells with 20 µL of diluted markers/ladders (Use 5 µL of E-Gel marker plus 15 µL of water). Continue loading 20 µL of sample per well.
  9. Electrophorese at 100V for 40 min. Make sure that the current is running in the correct direction.
  10. After the run, remove the gel with tray and visualize PCR fragment bands using the E-Gel Imager or other blue light source (Safe Imager 2.0 or E-gel Safe Imager)