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    Lab Manual GMB

    Uploaded by

    Deepika Choudhary
    1. The document describes the steps to perform agarose gel electrophoresis to separate DNA fragments by size. 2. Key materials needed include an electrophoresis chamber, gel casting trays, sample combs, TAE or TBE buffer, loading buffer with tracking dyes, and ethidium bromide stain. 3. The procedure involves casting the agarose gel, loading samples and a ladder, running electrophoresis, and visualizing bands under UV light.

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    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd

    Lab Manual GMB

    Uploaded by

    Deepika Choudhary
    108 views3 pages
    1. The document describes the steps to perform agarose gel electrophoresis to separate DNA fragments by size. 2. Key materials needed include an electrophoresis chamber, gel casting trays, sample combs, TAE or TBE buffer, loading buffer with tracking dyes, and ethidium bromide stain. 3. The procedure involves casting the agarose gel, loading samples and a ladder, running electrophoresis, and visualizing bands under UV light.

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    1. The document describes the steps to perform agarose gel electrophoresis to separate DNA fragments by size. 2. Key materials needed include an electrophoresis chamber, gel casting trays, sample combs, TAE or TBE buffer, loading buffer with tracking dyes, and ethidium bromide stain. 3. The procedure involves casting the agarose gel, loading samples and a ladder, running electrophoresis, and visualizing bands under UV light.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    108 views3 pages

    Lab Manual GMB

    Uploaded by

    Deepika Choudhary
    1. The document describes the steps to perform agarose gel electrophoresis to separate DNA fragments by size. 2. Key materials needed include an electrophoresis chamber, gel casting trays, sample combs, TAE or TBE buffer, loading buffer with tracking dyes, and ethidium bromide stain. 3. The procedure involves casting the agarose gel, loading samples and a ladder, running electrophoresis, and visualizing bands under UV light.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
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    Experiment no.

    1
    To perform Agarose gel electrophoresis Materials required:

    An electrophoresis chamber and power supply Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.

    Sample combs, around which molten agarose is poured to form sample wells in the gel. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.

    Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling.

    Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromidestained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light.

    Procedure 1. For a 1% agarose gel, weigh out 1g of agarose into a flask and add 100ml of 1 x TBE or TAE. Heat solution in a microwave or on hot plate until agarose is completely dissolved and gives a clear solution. 2. Allow to cool until it has a temperature of 50 55 C; for 10 min (approx.). 3. Prepare gel casting tray by sealing ends of gel chamber with tape or appropriate casting system. Place appropriate number of combs in gel tray. 4. Add 5 l of ethidium bromide to cooled gel and pour into gel tray. Allow to cool for 15-30 min at room temperature. Gels can also be placed in a cold space and used the following day. 5. Remove comb(s), place in electrophoresis chamber and cover with buffer (TAE or TBE as used previously).

    6. Add loading buffer to samples. As a guideline, add 1.5 l of 10x Loading Buffer to a 20-25 l PCR/DNA solution. For more concentrated DNA solutions (e.g. plasmids), prepare tubes with 8 l of 2x Loading Buffer and 2 l of plasmid. 7. Load DNA and standard (Ladder) onto gel. 8. Electrophorese at 100V for 1 h. 9. Visualize DNA bands using UV transilluminator or gel imaging system.

    Reagents/Solutions

    50 x TAE : 242g Tris base, 57.1 ml of glacial Acetic acid, 100ml of 0.5M EDTA (pH 8.0), Make up to 1 L with water. (set pH of EDTA with NaOH pellets; do not use HCl to alter the pH)

    5 x TBE: 54g Tris base: 27.5g of boric acid: 20ml of 0.5M EDTA (pH 8.0), Make up to 1 L with water.

    Trouble - shooting:
    If you see faint or no bands on the gel:

    There was insufficient quantity or concentration of DNA loaded on the gel. Increase the amount of DNA, but don't exceed 50 ng/ band.

    The DNA was degraded. Avoid nuclease contamination. The DNA was electrophoresed off the gel. Electrophorese the gel for less time, use a lower voltage, or use a higher percent gel.

    Improper W light source was used for visualization of ethidium bromide-stained DNA. Use a short wavelength (254 nm) W light for greater sensitivity. Note: For preparative gels, using a longer wavelength (312 nm) W light will minimize DNA degradation.

    If you see smeared DNA bands:


    The DNA was degraded. Avoid nuclease contamination. Too much DNA was loaded on the gel. Decrease the amount of DNA. Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30 C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity. This is done by checking the pH in the anode and cathode chambers.

    There was too much salt in the DNA. Use ethanol precipitation to remove excess salts, prior to electrophoresis.

    The DNA was contaminated with protein. Use phenol extractions to remove protein prior to electrophoresis.

    Small

    DNA

    bands

    diffused

    during

    staining.

    Add

    the

    ethidium bromide during electrophoresis. If you see anomalies DNA band migration:

    Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30 C during electrophoresis. Check that the electrophoresis buffer used had sufficient buffer capacity.

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