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Electrophoresis

Equipment. How Does it Work?. During electrophoresis, molecular fragments migrate through agarose, a Jello-like substance derived from seaweed, when exposed to electric current. . Negatively charged substances will move towards the positive electrode and positively charged substances will move towards the negative electrode. The gel-box serves as an electrical bridge between two submerged electrodes in a conducting solution..

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Electrophoresis

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    1. Electrophoresis

    2. Equipment

    3. How Does it Work?

    4. Negatively charged substances will move towards the positive electrode and positively charged substances will move towards the negative electrode. The gel-box serves as an electrical bridge between two submerged electrodes in a conducting solution.

    5. DNA is Negatively Charged

    6. As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. Voltage

    7. Buffers Several different buffers have been recommended for electrophoresis of DNA. 1. TAE (Tris-acetate-EDTA) 2. TBE (Tris-borate-EDTA) Buffers not only establish a pH, but provide ions to support conductivity.

    8. The movement of DNA in a gel is the result of 2 factors: charge and size.

    9. Casting the Gel

    13. Dyes for this lab will be loaded in the middle of the gel.

    15. DNA Learning Center Electrophoresis Animation http://www.dnalc.org/ddnalc/resources/electrophoresis.html

    16. Electrophoresis of Dyes

    20. By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs (2% for 0.2-1kb) Lower concentrations of agarose facilitate separation of larger DNAs (0.7% for 5-10kb) Agarose Concentration

    21. . The larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane

    22. You want to be able to see the DNA bands under UV light using SYBR Green. Typically, a band is easily visible if it contains about 20ng of DNA. We will load approximately 0.5 mg of DNA into each well. How Much DNA to Load?

    24. Marker Dyes Bromophenol blue migrates at a rate equivalent to 200–400bp DNA. Xylene cyanol migrates at approximately 4kb equivalence.

    26.

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