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    Lab5 (1)

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    alooshmaroosh1011

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    Lab5 (1)

    Uploaded by

    alooshmaroosh1011
    1 views3 pages

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    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    1 views3 pages

    Lab5 (1)

    Uploaded by

    alooshmaroosh1011

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 3

    Lab5 Experiment

    Experiment: Agarose Gel Electrophoresis

    Objective

    To separate DNA fragments by size using agarose gel electrophoresis and to visualize the separated
    DNA using a DNA stain.

    Materials and Reagents

    Gel Preparation

     Agarose powder
     Electrophoresis buffer (e.g., 1X TAE or 1X TBE)
     Microwave or hot plate for heating
     Casting tray and gel comb

    Sample Preparation

     DNA samples (e.g., PCR products, restriction digests)


     DNA ladder (size marker)
     Loading buffer (e.g., 6X loading dye)
     Microcentrifuge tubes and pipettes

    Staining and Visualization

     Ethidium bromide (EtBr)


     UV transilluminator or gel documentation system

    Equipment

     Electrophoresis chamber and power supply


     Gel casting tray and gel comb
     Microwave or hot plate
     Pipettes and tips

    Procedure

    1. Gel Preparation

    1. Determine Gel Concentration:


    o For standard DNA fragment separation, prepare a 1% agarose gel.
    o Adjust the agarose concentration if needed (e.g., 0.8% for larger fragments, 1.5% for
    smaller fragments).
    2. Prepare Agarose Gel:
    o Measure 1 gram of agarose powder and add it to 100 mL of 1X TAE or 1X TBE
    buffer.
    o Heat the mixture in a microwave or on a hot plate until the agarose is completely
    dissolved. Allow it to cool to about 60°C.
    3. Pour Gel:
    o Pour the melted agarose into a gel casting tray with a comb in place.
    o Allow the gel to solidify at room temperature (approximately 20-30 minutes).

    2. Sample Preparation

    1. Prepare DNA Samples:


    o Mix your DNA samples with 6X loading buffer in a microcentrifuge tube. The
    typical ratio is 1:5 (1 part loading buffer to 5 parts DNA sample).
    2. Prepare DNA Ladder:
    o Mix the DNA ladder with the loading buffer in a similar manner as the DNA
    samples.

    3. Setting Up Electrophoresis

    1. Prepare the Electrophoresis Chamber:


    o Place the solidified agarose gel into the electrophoresis chamber and cover it with
    1X TAE or 1X TBE buffer. Ensure the gel is fully submerged.
    2. Load Samples:
    o Using a pipette, carefully load the DNA ladder into the first well of the gel.
    o Load the DNA samples into the remaining wells of the gel.
    3. Run the Gel:
    o Close the electrophoresis chamber lid and connect it to the power supply.
    o Run the gel at a constant voltage (typically 80-120V) until the dye front has
    migrated an appropriate distance through the gel (usually 30-60 minutes).

    4. Staining and Visualization

    1. Stain the Gel:


    o After electrophoresis, carefully remove the gel from the casting tray.
    o Stain the gel with Ethidium Bromide (EtBr)
    o For EtBr, soak the gel in a 0.5 µg/mL solution for 10-15 minutes, then rinse briefly
    in water.
    2. Visualize the Gel:
    o Place the stained gel on a UV transilluminator or gel documentation system.
    o Visualize and capture an image of the DNA bands. The DNA bands will appear as
    bright bands against a dark background.

    5. Analysis

    1. Compare Band Sizes:


    o Compare the bands in your DNA samples to the bands in the DNA ladder. The
    ladder provides size markers to estimate the size of your DNA fragments.
    2. Evaluate Gel Quality:
    o Check for clear, well-resolved bands. Smearing or distorted bands can indicate
    problems with sample quality, gel preparation, or running conditions.

    Homework: what is the difference between TBE and TAE buffer?

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