推荐：PCR从引物设计到反应条件的优化

这份资料以言简意赅的幻灯形式展示了PCR从引物设计到反应条件的优化应注意的问题，相信对您的实验有指导意义。
极力推荐！
http://www.biotechlab.nwu.edu/pe/

包括以下内容：
Introduction - PCR Primer

Components
Primer Design

Primer Design - I

Primer Design - II

Melting Temperature, Tm

Tm Calculations, Nearest Neighbor Analyses I

Tm Calculations, Nearest Neighbor Analyses II

Quantitating Nucleic Acids

Calculating Oligonucleotide Molarity

Reaction Conditions-Background

Conditions Affecting Fidelity

Reaction Parameters That Affect PCR Efficiency

Conditions Affecting Fidelity

Reaction Set Up

Optimization

Optimization of PCR Reactions - Why?

Biochemical Reminder

Buffer Optimization

PCR Primer Concentrations
Template

PCR Product Copy Number Depends on Target Starting Copy Number

Input Copy Number, Number of Cycles, and Product Mass

Mg++

Mg++ in PCR

Mg++ Optimization

Enzyme

Enzyme Choice

Different PCR Enzymes for Different Applications

Different PCR Enzymes Mean Different PCR Buffers

Optimum Mg++ Ranges Depend Upon Polymerase

Effect of [Mg++] and [Enzyme] on Yield

Other Reaction Parameters and Potential Artifacts

The Plateau Effect

The Plateau Effect - I

Plateau Effect - II

Other Artifacts

Three-way competition in PCR

XX Mispriming Models

Pre-PCR Mispriming

Solution to Artifacts: Hot Starts

Hot Start Procedures To Increase PCR Specificity

Manual Hot Start Theory

Manual Hot Start Method

AmpliWaxTM Hot Start Method

Hot Start Enzymatic

Activation Curve of AmpliTaq Gold

Using a Pre-PCR Hold for Activation

Manual Hot Start Versus AmpliTaqTM Gold