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

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

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

Real Time PCR

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!!!

Detection of Product in Real Time

SYBR GREEN

Other Probes: TaqMan Molecular Beacon

CYCLE NUMBER 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

AMOUNT OF DNA 1 2 4 8 16 32 64 128 256 512 1,024 2,048 4,096 8,192 16,384 32,768 65,536 131,072 262,144 524,288 1,048,576 2,097,152 4,194,304 8,388,608 16,777,216 33,554,432 67,108,864 134,217,728 268,435,456 536,870,912 1,073,741,824 1,400,000,000 1,500,000,000 1,550,000,000 1,580,000,000

1600000000
AMOUNT OF DNA

1400000000 1200000000 1000000000 800000000 600000000 400000000 200000000 0 0 5 10 15 20 25 30 35 PCR CYCLE NUMBER

10000000000 1000000000 100000000 10000000 1000000 100000 10000 1000 100 10 1 0 5 10 15 20 25 30 35 PCR CYCLE NUMBER

AMOUNT OF DNA

Effect of Template Concentration

Making a Standard Curve