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DNA extraction

DNA extraction. recap. Safety point. Spill phenol on bare feet (flip flops!) - goodbye skin Spill phenol on shoes – maybe stained shoes Spill phenol on bare legs (shorts) - goodbye skin Spill phenol on pants – stained pants. Calculation of DNA concentration.

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DNA extraction

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  1. DNA extraction recap

  2. Safety point • Spill phenol on bare feet (flip flops!) - goodbye skin • Spill phenol on shoes – maybe stained shoes • Spill phenol on bare legs (shorts) - goodbye skin • Spill phenol on pants – stained pants

  3. Calculation of DNA concentration • Measure UV absorption spectrum

  4. Calculation of DNA concentration • Absorbance coefficient for dsDNA at 260nm = 50 • So DNA with an absorbance of 1 at 260nm = 50ug/ml (or 50 ng/ul) • We diluted your DNA 1 in 100 • Multiply the A260 by 50 to get concentration of diluted sample • Then multiply by 100 to get concentration of your sample

  5. DNA extraction from plants • Smush (“homogenize”) the cells • Dissolve membranes and solublize DNA in a detergent buffer. • Remove solids with centrifugation • Remove proteins with KOAc • Remove proteins with phenol extraction • Precipitate DNA with alcohol

  6. DNA extraction issues • Homogenization - has to be “just right” • RNA • Hydrodynamic shearing - treat it gently

  7. Agricultural biotechnology A major application of genetic engineering

  8. Plant genetic transformation • Need to engineer genes into a “vector” (recombinant DNA or genetic engineering) • Need to get vector into plant cells (gene delivery) • Need to grow whole plant from a single cell (regeneration)

  9. Gene Delivery Agrobacterium Particle gun

  10. Introduction of the gfp gene into different target tissues Petunia petal - cells Soybean seed Wheat callus - cells

  11. Plant Recovery Starting MaterialImmature seeds Soybean Regeneration Germination Induction Development Proliferation

  12. Cotton Xmas Cactus Fern Ohio Buckeye Soybean Poplar

  13. Biotech traits • “Input traits” • Make product easier or cheaper to produce • Herbicide resistance (e.g. Roundup Ready, Liberty Link). Labor saving, can reduce overall herbicide usage. • Insect resistance (e.g. many Bt products). Reduces or eliminates pesticide usage. • “Output traits” • Add quality to product. • Storage properties (e.g. Flavr Savr). Longer lasting fruit, easier to transport. • Composition (e.g. pharming, oil / protein).

  14. Bt Corn Reduces:Insecticide MycotoxinApplication Production

  15. Monarch larvae on Butterfly weed X

  16. Acreage of GMO Varieties

  17. Acreage of GMO Varieties

  18. Public Support Varies for Different Applications of Biotechnology(Includes ALL Countries – N = 35,000) Source: Thomas Hoban, North Carolina State University

  19. The Benefits of Using Biotechnology are Greater than the Risks (35,000 Consumers from 35 Countries)

  20. The Benefits of Using Biotechnologyare Greater than the Risks (European Consumers)

  21. Willing to Buy GM Food if More Nutritious (Net = Continue – Not Continue)

  22. Perceptions of safety improved when US consumers learn that biotech foods have been in the stores. Do you think genetically modified foods are basically safe, basically unsafe, or don’t you have an opinion on this? Now, as you know, more than half of products in the grocery store are produced using some form of biotechnology or genetic modification. Knowing this, do you think genetically modified foods are basically safe, basically unsafe, or don’t you have an opinion on this? January 2001 Source: Pew Initiative on Food and Biotechnology

  23. “By Eating a Genetically Modified Fruit, a Person’s Genes Could also Be Changed” 1996 - 1998 Source: Thomas Hoban, North Carolina State University

  24. “Ordinary Tomatoes Do Not Contain Genes, while Genetically Modified Ones Do” 1996 - 1998

  25. Thanks • Thanks to Monsanto for making MTU available • Thanks to John Finer for biotech images.

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