PCR引物设计

・ What's PCR? (Michael Blaber's Lab)Illustrated introduction to usr/localious aspects of PCR technique. It's very valuable not only for those new to PCR but also those familiar with PCR.・ ...

・ Agarose Gel Electrophoresis of PCR Products (Robert H. Cruickshank)・ Agarose Gel Electrophoresis of PCR Products (Immunology Resource)・ DIAPOPS Technique (Detection of Immobi ...

PCR Primer Design and Reaction Optimization (Molecular Biology Techniques Manual) PCR Primer Design (Newman Lab) PCR Primer Design (Eppendorf)Detailed guide for primer design. PCR Primer Design ...

・ Long PCR (Church Lab)PCR conditioning for different templates primer design and moreLong PCR Reagents and Guidelines (Harusr/locald)Detailed protocol for long distance PCR including template ...

About in situ PCR (Applied Biosystems)Basic information about in situ PCR and its applications. The In Situ PCR: Amplification and Detection in a Cellular Context (Ernest F. Retzel et al)General guide ...

・ Standard PCR Protocol (Molecular Biology Techniques Manual)The followings are described in detailRecommended Reagent ConcentrationsRecommended Reaction Conditions"Hot Start" PCRAsymmetric PC ...

主要内容如下：・ RT-PCR ・ Competitive and Quantative RT-PCR ・ In Situ RT-PCR ・ RL-PCR ・ DNA Contamination ・ RT-PCR FAQ RT-PCR・ RT-PCR Protocol (UMBC)Fir ...

・ PCR Additives (Robert H. Cruickshank)A usr/localiety of PCR additives and enhancing agents have been used to increase the yield specificty and consistency of PCR reactions. Whilst these addi ...

Recommended Reagent Concentrations: Primers: 0.2 - 1.0 uM Nucleotides: 50 - 200 uM EACH dNTP Dimethyl sulphoxide (DMSO): 0 - 10% (v/v) Taq polymerase: 0.5 - 1.0 Units/50ul rxn Target DNA: 1 ng - 1 ug ...

Extrachromosomal Elements-Plasmids (Michael Blaber)What's plasmid? Find answer here. It provides background information on the features of plasmid selection marker and more... Rescuing Integrated ...

Recommended Reaction Conditions: Initial Conditions: Initial denaturation at start: 92 - 97oC for 3 - 5 min. If you denature at 97oC denature sample only; add rest of mix after reaction colls to annea ...

Labelling PCR Products with Digoxigenin PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concen ...

"Hot Start" PCR: In certain circumstances one wishes to avoid mixing primers and target DNA at low temperatures in the presence of Taq polymerase: Taq pol is almost as efficient as Klenow pol at 37oC; ...

Cloning PCR Products T-A Cloning Strategy: Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated addition of a single nucleotide to the 3'-ends of P ...

・ Plasmid Midi-preps (NWSFC)Diatomaceous Earth-based midi-prep. This procedure is the method of choice for isolating double stranded plasmid-based templates for the Sequenase Dye-Labeled Termi ...

・ Standard (alkaline lysis) Mini-Prep (Goldberg Lab)Standard protocol for mini-prep and recipe for solution I II and III.Alkaline Lysis Minipreps of Plasmid or Cosmid DNA (Donis Keller Lab)Pla ...

・ Plasmid Mini and Maxi Prep Methods (Gimila Lab) ・ Maxi-preps and all media solutions (NWFSC)Isolation of cosmid plasmid and P1 DNAs via diatomaceous earth-based Maxi Midi and Mini-p ...

Preparation of Cosmid DNAPreparation of Cosmid DNA from 50 ml Cultures (Donis Keller Lab)Cosmid vectors containing foreign DNA inserts are known to replicate more slowly than small plasmids in culture ...

把PCR产物克隆到载体上常见有3种做法：1.直接克隆到T载体（或者U载体上）2.引物设计时引入酶切位点，PCR产物酶切后直接克隆到目标载体上3.将平末端PCR产物直接克隆到平末端酶切载体上。 方法3主要是针对高保真的聚合酶比如Stratagene公司的Pfu酶，New England Biolabs公司的Vent酶，以及QIAGEN公司新出的同时具有热启动功能的高保真ProofStart酶 ...

PCR条件的优化是一个麻烦耗时的过程，涉及到温度、时间、镁离子、引物、dNTP、Taq酶、模板等多个因素的调整。一般来说利用热启动，比如Platinum Taq DNA聚合酶（Invitrogen）可以达到更高的特异性，降低对镁离子浓度的依赖，并且有利于提高“问题模板”的产量。然而，传统的PCR条件优化策略经常对含有稳定二级结构的DNA序列无效，因为这些序列难以变性，并且在退火过程中影响引物与模板 ...