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Quantitative Techniques

Quantitative Techniques. CE 742: Advanced Topics in Environmental Engineering. Epiflourescence Microscopy. Epifluorescence Microscopy. Produces UV light. Reflects UV light (other wavelengths pass). Molecules excited by UV emit fluorescent light. FISH Images.

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Quantitative Techniques

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  1. Quantitative Techniques CE 742: Advanced Topics in Environmental Engineering

  2. Epiflourescence Microscopy

  3. Epifluorescence Microscopy Produces UV light Reflects UV light (other wavelengths pass) Molecules excited by UV emit fluorescent light

  4. FISH Images Comparison of Conventional Microscopy with Fluorescence microscopy- species differentiation of Achromatium cells living in sediment. Microbial Ecology Group, University of Newcastle.

  5. FISH Image EXAMPLE: • The red cells are Archaea from the ANME-2 group • The green stained cells are sulfate-reducing bacteria related to the Desulfosarcina. Photo courtesy of Victoria J. Orphan, Caltech, Pasadena CA

  6. Real Time PCR

  7. Real Time PCR • PCR • most sensitive technique available • Two primers means high • technically simple • but difficult to get truly quantitative results using conventional PCR • Real-Time PCR • All of the benefits of PCR with ability to Quantify!!!

  8. Detection of Product in Real Time SYBR GREEN Other Probes: “TaqMan” “Molecular Beacon”

  9. Effect of Template Concentration

  10. Making a Standard Curve

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