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Agarose Gel Electrophoresis Notes

Agarose Gel Electrophoresis

Overview

  • Gel electrophoresis separates DNA fragments based on size (number of base pairs) using a solid support medium like agarose gel.
  • DNA samples are pipetted into wells, and an electric current is applied, causing the negatively charged DNA to migrate towards the positive (anodal) end.
  • Migration rate is inversely proportional to size: smaller fragments move faster.
  • The charge in electrophoresis:
    • Anode: positive pole.
    • Cathode: negative pole.

Agarose Gel Electrophoresis System

  • The system includes supplies and accessories needed for AGE.

  • DNA particles are separated based on their charge.

    • DNA is negatively charged.
    • Opposites attract:
      • Positive particles attract negative particles.
      • Negative particles attract positive particles.

  • Electrode Orientation:

    • Cathode is the negative pole (black electrode).
    • Anode is the positive pole (red electrode).

  • Principle of AGE: Opposites attract allows separation of particles.

    • A negatively charged sample in the well migrates towards the anode when voltage is applied (DNA runs from black to red).
    • Incorrect orientation causes reverse migration.

  • DNA fragments are separated based on size during migration.

    • Low molecular weight (smaller fragments) move faster towards the anode.
    • High molecular weight (larger fragments) move slower.

Parts of AGE

  • Power Supply: Source of electricity.
  • Gel: Where nucleic acid migrates towards the electrodes.
  • Electrophoretic Buffer (Running Buffer): TAE and TBE.
    • TAE: good for 100 bp to 1 kb fragments.
    • TBE: good for smaller fragments.
  • Loading Wells: Where DNA samples are dispensed.
  • AGE Chamber/Tank

Accessories

Gel

  • Solidified agarose (purified linear galactan hydrocolloid derived from agar).
  • Contains pores for particle movement toward the oppositely charged side.
  • Poured as a hot liquid and solidifies upon cooling.

Casting Tray

  • Mold for the gel where dissolved agarose solution is poured.

Comb

  • Well-former template placed across the casting tray.
  • Contains teeth that create the wells.
  • Comb size varies; sample volume depends on comb size.

Wells

  • Formed when hot gel solidifies around the comb's teeth.
  • Provide a place for samples.
  • Number of wells depends on the work being done and gel size.

Gel Electrophoresis Chamber/Tank

  • Where the running buffer is poured, and the gel is submerged.
  • Used for analysis and size separation of nucleic acids.
  • Size depends on the brand and model.

Gel Doc and/or UV Transilluminator

  • Imaging instruments for recording and analyzing gel electrophoresis results.
  • Equipped with software to store and transfer samples via USB.

DNA Ladder

  • Also known as a molecular marker.
  • Reference to estimate the size of unknown DNA molecules.
  • Typically placed in lane 1.
  • Ladder increments can be 100 bp or 1 kb, depending on the size range.

Loading Dye

  • Weighs down the sample in the solution to prevent it from leaving the well.
  • Includes a visible marker to track the run's progression.
  • Incorrect sample orientation means loading was done on the opposite side.

Gel Stain

  • Solutions used to visualize separated protein bands or nucleic acids.
  • Examples: Ethidium-Bromide, SYBR green stains, Cyber green.
  • GelRed is a safer alternative.

Reagents

  • 1X TAE Buffer
  • Molecular Biology Grade Sterile Water or Double Distilled Water
  • Agarose
  • 6X Loading Dye
  • DNA Ladder - 100bp
  • Gel Red Stain 10,000X

Glassware/Plasticware

  • Analytical balance
  • Measuring cups
  • Spatula spoons
  • Conical flask
  • Measuring cylinder
  • Beaker
  • Automatic pipet 0.5-10μL
  • Automatic pipet 10-100μL
  • Pipet tips
  • Microwave or hotplate
  • Vortex mixer
  • Agarose gel electrophoresis system
  • Tabletop microcentrifuge (with rotor for 2.0 mL tubes)
  • Gel documentation system
  • Parafilm or PCR tubes

AGE Procedure

Preparation of Buffer Solution: 1X TAE (500mL)

  1. Measure 490 mL of double distilled water or molecular grade sterile distilled water.
  2. Add 10 mL of 50X TAE buffer in the solution.
  3. Mix well
  4. Place in a 500 mL sterile reagent bottle
  5. Label accurately, including the date of preparation.
  • (C1V1)(C2V2)

Preparation of 50mL 1% Agarose Gel

  1. Measure 50 mL of 1X TAE in conical flask
  2. Add 0.5 g of agarose gel
  3. Dissolve the agarose by heating in a microwave or on hot plate
  4. Gently swirl the glass occasionally (ensure that the lid of the flask is loose to avoid buildup of pressure)
  5. Check until the solution is clear and completely dissolves.
  • Agarose gel concentration determines pore size in the gel matrix.
  • Pores serve as sieves that allow movement of nucleic acid.
  • Lower fragment size and concentration result in faster DNA movement.
  • Higher fragment size and concentration result in slower DNA movement.
  • Concentrations typically range from 0.7% to 2%.
  • 0. 7% is good for large, high molecular weight fragments (fragile).
  • 2% is good for fragments around 200bp-1kb in size.
  • 3% the gel is brittle.

Staining of Agarose Gel

  1. Dilute the GelRedTM 10,000X stock reagent into the agarose gel solution at 1:10,000
  2. For 50 mL melted agarose, add 5 μL GelRedTM
  3. Gently swirl and make sure that the dye is thoroughly mixed with the solution.
  4. Cast the gel once it has cooled down.
  • Add when agarose dissolved and cooled but can be added while hot if manufacturer instructions allow.

Preparation of Casting Tray

  1. Attach the rubber dams or use a tape to tape the ends of the casting tray to create the end walls.
  2. Secure the tapes by taping over the ends of the tray.
  3. Select comb depending on the number of samples you will run
  4. Place well forming comb in the groove toward the end of casting tray
  5. Adjust the level of comb evenly and ensure leaving a few mm space from the teeth of comb and tray.
  6. Place the casting tray on a level surface.

Casting of Agarose Gel

  1. Slowly pour the cooled melted agarose in the assembled casting tray without creating bubbles in the gel.
  2. Use a pipet tip to push or remove any bubbles to end of the casting tray.
  3. Pour enough melted agarose until it reaches to 3 - 5 mm thickness.
  4. Do not fill the casting tray to the top
  5. Allow the gel to sit undisturbed for at least 20 minutes until it is firm. 60 minutes is the best time to solidify.
  6. Gel is completely firm if it has turned cloudy and opaque.
  7. Remove the tapes from the ends of the tray.

Addition of the Running Buffer and Comb Removal

  1. Prepare the electrophoresis chamber
  2. Carefully pour 300 mL (or up to the indicated level mark) of the previously prepared 1X TAE running buffer in electrophoresis tank reservoir.
  3. Carefully remove the comb with both hands by gently and carefully lifting them upwards.
  4. Place the previously casted agarose gel on the gel platform in the middle of the electrophoresis chamber.
  5. Do not remove the gel from the gel (It is important to check the position of the gel, the wells must be on the cathode side and will run towards the anode "BLACK TO RED")
  6. The running buffer should completely cover your gel and must be fully submerged (Add additional buffer if gel is not fully submerged).

Loading of Samples

  1. Retrieve extracted DNA(remember to work on ice), prepare loading dye and the DNA ladder

  2. Prepare sample in either: (loading dye+samples)

    a. In the reaction tube that your sample is in
    b. In a new PCR tube
    c. Using parafilm (economically accepted and cheaper)
    d. 96 well plate

  3. Using a clean micropipette, dispense 2 uL of 6X (not universal this depends on the protocol) loading dye in any of the vessel mentioned above

  4. Add 10 uL of DNA extract to each loading dye and pipette mix several times

  5. Add 2 uL of the DNA ladder to the first well (DO NOT ADD LOADING DYE).

  6. Dispense by slowly pipetting your stained/dyed samples one by one into each well

Running the Gel

  1. Place the lid of the electrophoresis chamber
  2. Make sure that the safety bars fit correctly into the slits of the power supply
  3. Connect the electrodes to the power supply. Make sure the positive and negative electrodes are properly connected, the black lead to the negative terminal and red to the positive terminal.
  4. Turn on the power supply and adjust the voltage to 50-100 Volts (5 to 10 volts/cm of gel) and current (100V, 90mA)
  5. Run the agarose for 25 minutes or until the dye is 50%-60% of the way down the gel
  6. Turn off the power, remove electrodes and the lid
  • Voltage is important as it is the pressure that will push the electricity.
  • Current will help in the flow of electrons

Analysis of Gel

  • Remove the gel from the electrophoresis chamber
  • Slide the gel without the tray in the gel documentation system
  • The separated DNA will appear as distinct band/s on the gel
  • The gel can be photographed, or in the case of the gel documentation system the photo of the gel can be saved in the system
  • BANDS: horizontal bars of stained DNA molecules embedded in the gel and visualized under gel viewer.
  • The more concentrated the DNA the thicker and the brighter the band.
  • The bands are found above molecular marker because this is Genomic DNA or gDNA. gDNA contains very high molecular weight ( more than 10kb). Di bababa dahil masyadong mabigat kaya nag stastay near the negative area.