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Agarose gel electrophoresis

Written on 01.07.2011

Purpose

To analyse DNA composition of a sample. Voltage-dependent DNA migration through agarose gel allows sequences separation according to their size.

Ingredients & Materials

For a 1% agarose gel

Sample loading buffer will also be needed.

Materials: bottle, tray, cast, comb, electrophoresis chamber, voltage/current generator, pipette, microwave, weight, spoon, labcoat...

Alternative (with TAE buffer)

Loading samples

• To check the sizes of PCR products:

• • 3 ul ddH2O
  • 2 ul DNA
  • 1 ul loading dye 5x

• To separate your product and later cut it out of the gel:
  • 48 ul DNA (that is everything left from the 50 ul PCR tube)
  • 10 ul loading dye 6x

Method

• To prepare the gel:
  • • Weight agarose, place it into a bottle with a cap, add TBE 1x buffer
    • Place the bottle into a microwave, loosen the cap, heat it untill it almost boils and the agar is dissolved
    • Cool it down until it can be hold (that can be done under water)
    • Add 1.25µl of GelRed
    • Pour into the casting tray (it is easy to assemble the casting but a way to difficult to discribe), put the comb and wait until it polymerizes
• To run the gel:
  • Take out the comb and place the tray with your gel into the electrophoresis chamber (usually already filled with buffer) so that your wells are nearer to the cation, red side.
  • TBE 1x buffer should be covering the tray, add more if needed
  • In a PCR tube mix ~10µl of your sample with 1.5µl of loading solution (quantities indicated may vary with the sample concentration )
  • Load about 5µl of your sample (this quantity varies with the thickness of the comb used and DNA concentration) and DNA ladder onto gel, taking care not to diffuse it and not to displace the gel
  • Place the cover, red wire to red side and inversely, plug it into the tension/current generator
  • On the generator set for voltage: constant mode 80V and time: 60min (faster run with higher voltage as for example 150V may affect the quality of bands separation)
  • After run is over, take the tray out on some plate, and bring it to visualize DNA bands to a UV lightbox or gel imaging system
  • If you are the first to use the computer, log in as guest (no password needed)
  • Gel can be disposed in a normal biological waste or kept covered in an aluminum foil