How do I Line Up DNA for Analysis?

Agarose gel electrophoresis is the most common method of visual DNA fragment analysis, which allows technicians to visually discern DNA fragments based on size. The agarose gel sustains a magnetic field and, since DNA has a negative charge, it moves towards the positive end of the magnetic field. Smaller DNA fragments move more quickly through the gel and larger fragments move more slowly. DNA fragments are fluorescently stained with an intercalating agent known as ethidium bromide. This allows for comparison of several electrophoresed DNA samples on a gel.

Things You'll Need

• Agarose gel, 0.7 percent to 2 percent
• Tris-borate-EDTA (TBA), tris-acetate-EDTA (TAE), sodium lithium acetate (LA), boric acid (SB) buffer
• Ethidium bromide
• Gel loading dye
• Gel tray
• Electrophoresis chamber
• Gloves
• Protective eye wear
• Pipette
• Electrophoresis comb

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Instructions

Preparing a 0.7 Percent Agarose Gel

• 1

  Weigh out 0.7 grams of agarose powder and then place into a large (250 ml) conical flask.

• 2

  Add 100ml of a 1X (regular strength) buffer (TAE, TBE, SB or LB buffer) to the conical flask containing the agarose powder.

• 3

  Microwave or heat using a bunsen burner for approximately one minute until the solution becomes clear and the agarose has dissolved.

• 4

  Remove the flask from the heat source and allow it to cool to 55 to 60 degrees Celsius.

• 5

  Add 1 µL (microliter) of ethidium bromide to the cooled agarose solution using an appropriately sized pipette, usually 1ml. Swirl the solution to mix.

• 6

  Pour the gel slowly into the gel tray, ensuring there is no bubble formation during this process. If there are no supplied well dams with the gel tray, use masking tape to form them.

• 7

  Place an electrophoresis comb carefully into the gel, ensuring a firm position and in the correct orientation. Electrophoresis combs can vary greatly on the number of teeth they have. Allow the gel to set for approximately 25 minutes.

• 8

  Submerge the gel in the same buffer used to prepare the agaorse solution--in this case, a 1X buffer. Submerge the gel in up to 5 ml of running buffer.

Preparation and Loading of DNA into the Agarose Gel for Analysis

• 9

  Transfer 5 to 10 µL of your sample(s) from their reaction tubes to fresh tubes. In the case that you want to use the entire reaction mixture, a fresh tube is not necessary.

• 10

  Add 0.2X loading buffer to each of your fresh sample tubes containing your DNA sample for loading.

• 11

  Remove the electrophoresis comb slowly, ensuring that you do not break the gel or create any space between the bottom of the gel and the gel tray.

• 12

  Add five to 12 µL of an appropriate DNA marker to the first well created by the electrophoresis comb. Whether or not a DNA marker is appropriate depends upon the size of the fragments you are expecting from your sample.

• 13

  Load five to 10 µL of your samples into the remaining wells and add an equal amount of the same DNA marker into the final well.

• 14

  Place the electrodes into the electrophoresis chamber by plugging the wires into the correct sockets into the chamber and set the electrode to 50 to 150 V.

• 15

  Allow the gel to run for one to four hours.

Analysis of Results

• 16

  Remove the gel from the gel chamber and place it either in a UV room for visual identification, or place into a machine that is able to take a photograph of the gel.

• 17

  Measure the markers distance of migration in their wells.

• 18

  Plot the distances against the size of the bands on semilog graph paper and draw a best fit line.

• 19

  Calculate the distances of your unknown bands.

• 20

  Estimate the sizes of your unknown bands by drawing a line up from the distance traveled by your unknown bands that meets the line of best fit.

• 21

  Draw another line from this meeting point over to the size axis.