Manipulating DNA and RNA. DNA hybridization PCR.

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Presentation transcript:

Manipulating DNA and RNA

DNA hybridization

PCR

PCR Movie

PCR Applications

PCR Applications Forensic Science

DNA Cloning

DNA cloning and amplification

Protein Production by DNA Cloning

Gene Mutation

Digestion and Destroy

Localization or Amplification of proteins by DNA Cloning

Detection of protein interactions by DNA Cloning

Yeast two-hybrid system

Identify Tumor Biomarkers by DNA Cloning Phage display

Northern and Southern Blots RNA and DNA Detection

Northern and Southern Blots

Microarray

SDS and b-mercaptoethanol for Electrophoresis

SDS PAGE I

SDS PAGE II

SDS PAGE III

Western Blot

Visualizing Cells

Resolving Power

Light Microscope

Interference between light waves

Resolution Calculation

Two ways to get contrast

Four Types of light microscopy Bright field, phase contrast, differential interference contrast, Dark-field microscopy

Fluorescence Microscope

Fluorescent Dyes

Fluorescent image Blue: DNA; Green: microtubules; Red: centrimere

Immunofluorescence

Immunostaining

Confocal Fluorescence Microscopy

The difference between conventional and confocal microscopes

3D reconstruction from confocal images

Transmission Electron microscopy (TEM, resolution nm)

A root-tip cell under electromicroscopy

The scanning electron microscope (SEM)

Stereocillia from a hair cell

Live Cell Imaging

Green Fluorescence Protein (GFP)

Jellyfish and GFP Osamu Shimomura Noticed GFP in 1962

Dr. Douglas Prasher

Martin Chalfie

Sergey A. Lukyanov The Discovery of DsRed

Roger Y. Tsien

Track Molecular Motions

Passive Applications of GFP GFP-microtubules GFP-Actin CFP-Actin/YFP-Microtubules GFP-Actin

Spy on their Actions! FRET

The Principle of Fluorescence Resonance Energy Transfer (FRET) When the fluorophores are far apart: No FRET ExcitationEmission When fluorophores are close: FRET occurs ExcitationEmission FRET

FRET-Based Biosensors Calcium Ras and Rap1 Miyawaki, et al 1997, Nature Mochizuki, et al 2001, Nature Ting, et al 2001, PNAS Tyrosine Kinases

Design Strategy Weak FRET Phosphatase Strong FRET 433 nm 527 nm 433 nm 490 nm ECFP (1-227) SH2 (from c-Src) SubstrateEYFP Linker Src Activation

The Src reporter with CFP and YFP monomers ECFP (1-227) SH2 (from c-Src) SubstrateEYFP Linker A206K