• Manipulating DNA and RNA

DNA hybridization

PCR

PCR Movie

PCR Applications

PCR Applications

PCR Applications Forensic Science

DNA Cloning

DNA cloning and amplification

Protein Production by DNA Cloning

Gene Mutation

Gene Mutation

Digestion and Destroy

Localization or Amplification of proteins by DNA Cloning

Detection of protein interactions by DNA Cloning

Detection of protein interactions by DNA Cloning

Detection of protein interactions by DNA CloningYeast two-hybrid system

Identify Tumor Biomarkers by DNA CloningPhage display

Northern and Southern BlotsRNA 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

Western Blot

• Visualizing Cells

Resolving Power

Light Microscope

Interference between light waves

Resolution Calculation

Two ways to get contrast

Four Types of light microscopyBright field, phase contrast, differential

interference contrast, Dark-field microscopy

Fluorescence Microscope

Fluorescent Dyes

Fluorescent imageBlue: 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 0.002 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 ShimomuraNoticed 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

Excitation Emission

When fluorophores are close: FRET occurs

Excitation Emission

FRET

FRET-Based BiosensorsCalcium 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) Substrate EYFPLinker

Src Activation

The Src reporter with CFP and YFP monomers

ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker

A206K A206K

0.5

0.35