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Gel Electrophoresis

Gel Electrophoresis. Measurement of DNA Concentration. Agarose Gel Electrophoresis ( Bases for DNA separation) Agarose melted in a buffer and poured into a horizontal tray When solidified, the gel contains small holes or pores through which the DNA fragments will travel

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Gel Electrophoresis

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  1. Gel Electrophoresis Measurement of DNA Concentration

  2. Agarose Gel Electrophoresis (Bases for DNA separation) Agarose melted in a buffer and poured into a horizontal tray When solidified, the gel contains small holes or pores through which the DNA fragments will travel The percentage of agarose used to make the gel determines the ability of the gel to separate DNA fragments of different sizes Most common is 0.5–2% agarose Higher percentage will separate small DNA fragments better Lower percentage is better for separating large fragments

  3. Agarose Gel Electrophoresis To run a gel, it is submerged in a buffer solution that conducts electricity DNA is loaded into wells at the top of the gel Electric current is applied to the ends of the gel DNA migrates according to its charge and size Rate of migration through the gel depends on the size of the DNA Large fragments migrate slowly; smaller fragments migrate faster Tracking dye is added to the samples to monitor DNA migration during electrophoresis DNA can be visualized after electrophoresis by the addition of DNA staining dyes

  4. Agarose Gel Electrophoresis

  5. DNA Electrophoresis of uncut and cut plasmids C: Control (uncut)

  6. λ HindIII 1 2 3 4 5 6 7 • Agarose gel electrophoresis of different plasmids bp 23 130 9416 6557 4000 2322 2027

  7. Spectrophotometry • A method of chemical analysis based on the absorption or attenuation by matter of electromagnetic radiation of a specified wavelength or frequency • It is used to quantify the concentration of the absorbing substance (Beer-Lambert Law) • A material's absorption spectrum shows the fraction of incident electromagnetic radiationabsorbed by the material over a range of frequencies • Usually, the wave length at which the highest absorption takes place is used for measurements • A spectrophotometer is the device used for measuring light intensity as a function of the color, or more specifically, the wavelength of light.

  8. Determination of DNA Concentration • Measuring the absorption (Optical density, OD) of a DNA solution allows calculation of DNA concentration • At wavelength of 260 nm: DNA Conc = (OD x Dilution)/ Extinction coefficient ds DNA Conc = (OD x Dil ) / 20 is a measurement of how strongly a chemical species absorbs light at a given wavelength

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