Agarose Gel electrophoresis

This technique is used to separate DNA fragments by their size

Principle of Agarose Gel Electrophoresis

  • DNA loaded into one end of the gel

  • electric current applied to pull them through the gel

  • Due to the negative charge of the DNA fragments, they move towards the positive electrode

  • smaller fragments move through the gel faster than the larger ones

  • when the gel is stained with the DNA binding dye, the DNA fragments can be seen as bands

  • each band represents a group of same sized DNA fragments

materials required

  • An electrophoresis chamber and power supply

  • gel casting tray and comb

  • agarose gel

    • a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of red algae

    • melts - 85ºC , solidifies - 35ºC

    • Agarose polymer in solidified gel forms a porous network

      • pore size can be adjusted by varying the percentage of the agarose in the gel, in order to efficiently resolve nucleic acids of different sizes

      • lower agarose concentration (large pore sizes) used to separate larger DNA fragments

      • higher agarose concentration (smaller pore size) used to separate smaller DNA fragments - hence better separation and resolution of smaller DNA fragments

    • DNA staining agent is added into gel before it solidifies

  • electrophoresis buffer e.g. Tris-borate-EDTA (TBE)

    • molten gel is loaded into gel casting tray to set (the comb creates the wells

    • solidified gel is submerged in electrophoresis buffer

    • electrophoresis buffer is an ionic solution with buffering capacity

    • used in gel runs to allow current flow while impeding pH changes that may occur

  • Loading dye

    • contains a dense solution (e.g. glycerol)

      • mixed with DNA sample to allow the sample to “sink” into the wells

  • DNA staining agent e.g. SYBR Green, Ethidium bromide

    • inserts in between the bases of DNA stranf and fluoresce orange under UV → carcinogen

  • UV Transilluminator

how the machine works

  • electrophoresis chamber is connected to power pac

  • electric current causes DNA to move through gel

steps to conduct gel electrophoresis

  1. melt the agarose powder in TBE buffer

  2. add SYBR Green dye

  3. Pour the molten agar into a gel casting tray

  4. Use a comb to create the wells

  5. Allow the gel to set

  6. Submerge the gel in TBE buffer in an electrophoresis chamber

  7. Mix DNA sample with loading dye

  8. Load the DNA sample into the wells

  9. Connect to power supply

  10. View the gel with UV light

components and funcitons

power supply

  • provide the electric current for DNA fragments to move from negative to positive electrode

gel casting comb

  • make wells in the gel for loading DNA sample

agarose gel

  • forms a gel matrix with pore sizes for separation of DNA fragments

TBE buffer

  • allow current flow while impeding pH changes that may occur

Loading dye

  • contains glycerol which allow DNA sample to sink into wells

  • contains dye (bromophenol blue) which provides colours for easy monitoring of sample loading and progress of the electrophoretic run

DNA staining agent

  • allow for visualisation of DNA samples after the run under UV light

UV transilluminatior

  • to visualise and capture the gel image

analysing results

DNA marker / ladder

  • short fragments of DNA of known sizes (bp)

  • loaded together with the unknown DNA samples as reference

  • plasmids exhibit 3 conformations

    • when both strands are intact → supercoiled

    • when one strand is nicked → open circular

    • when both strands are cut → linear

more !!

  • different conformations of the same plasmid (linear, open circular, supercoiled) migrate at different speeds during agarose gel electrophoresis

  • both shape and molecular weight affect travelling distance

determination of DNA molecular weight

  1. Measure the distances travelled by 5 DNA marker bands around the DNA band and the DNA band

  2. Convert MW (molecular weight) of DNA marker bands into logarithm format (just log the MW) this is to make the graph linear

  3. plot graph of travel distance (mm) [y axis] vs logMW [x axis]

  4. use graph to determine the MW of the DNA of interest (rmb to un-log)