PCR技术

一、组织抽提：1. 取组织块50-100�用液氮在研钵中研磨成粉末2. 移入玻璃匀浆器加入1ml Trizol 抽打匀浆3. 移入1.5ml新离心管用5 ml针头抽打4. 冰上孵育5分钟5. 离心12000g 4℃ 5分钟 取上清液移入1.5ml新离心管6. 加0.2 ml氯仿，震荡，冰上孵育5分钟7. 离心0 ...

　PCR的假阳性问题深受重视，但我个人认为PCR假阴性问题相对于临床检测更为严重。其实PCR假阳性的问题是比较单纯的，一般仅涉及污染和引物的非特异性问题。而污染可以通过严格的实验室管理，合理的环境设置，以及加入UNG酶抗污染基本可以解决，而引物的非特异性问题基本属于厂家试剂质量问题。而PCR的假阴性却不同，它涉及了与PCR实验的几乎所有人员和技术环节，十分复杂。就临床而言，大三阳检出率低、甚至于G ...

The DNA Facility houses the “real-time” or kinetic PCR instrument the Applied Biosystems Model 7700 sequence detection system (the TaqMan instrument). The polymerase chain reaction (PCR) has revolutio ...

Why PrimerBank?PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). There are several ways to search for primers ...

PCR引物设计的11条黄金法则1.引物最好在模板cDNA的保守区内设计。DNA序列的保守区是通过物种间相似序列的比较确定的。在NCBI上搜索不同物种的同一基因，通过序列分析软件（比如DNAman）比对（Alignment），各基因相同的序列就是该基因的保守区。2.引物长度一般在15~30碱基之间。引物长度（primer length）常用的是18-27 bp，但不应大于38，因为过长会导致其延伸温 ...

Degenerate PCRby Michael Koelle 1/6/96Primer Design Graphic The identification of novel members of gene families by PCR using degenerate primers has been considered more of an art than a science so mu ...

Degenerate PCRby Michael KoelleThe identification of novel members of gene families by PCR using degenerate primers has been considered more of an art than a science so much so that the methods books ...

Degenerate Primers For amplification of cognate sequences from different organisms or for "evolutionary PCR" one may increase the chances of getting product by designing "degenerate" primers: these wo ...

IntroductionPolymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a rapid inexpensive and simple means of producing ...

The bisulfite reaction reaction was first described in early 1970s and was used by Frommer et al to distinguish between cytosine and 5mCytosine (5mC) in DNA. In this reaction DNA is first treated with ...

Bisulfite Treatment of DNAAdapted from Frommer et.al.*Dilute DNA (up to 2 mg) into 50 ml with distilled H2O. Add 5.5 ml of 2M NaOH. Incubate at 37°C for 10 minutes (to create single stranded DNA). Add ...

Methylated CpG Island AmplificationProtocol written by Minoru Toyota2. Materials2.1. MCARestriction enzymes SmaI XmaI T4 DNA ligase Taq DNA polymerase 10X PCR reaction buffer: 670mM Tris-HCl pH 8.8 40 ...

Primer Design Rules for Bisulfite Conversion Based PCRAuthor: Long-Cheng LiSource: Protocol OnlineAbstract: Primer design rules for MSP and bisulfite sequencing PCR. A. General consideration for pri ...

Methylation-Specific PCRProtocol written by James HermaundefinedMethylation-specific PCR (MSP) is a simple rapid and inexpensive method to determine the methylation status of CpG islands. This approach allo ...

Singel Nucleotide Primer Extension (SNuPE)Contributed by Dr. A. GratchevSingle Nucleotide Primer Extension is a powerful method which can be used for the precise analysis of methylation in a certain p ...

Bisulfite-PCR for Restriction Analysis and/or SequencingProtocol written by Jean-Pierre Issa based on several published papers.Bisulfite-PCR followed by restriction is a rapid and semi-quantitative me ...

TABLE OF CONTENTS 1. Abstract 2. Introductory statement 3. The key preparatory steps 3.1. Fixative 3.1.1. Protease digestion 3.1.2. Definition of optimal protease digestion 3.1.3. Definitions of subop ...

Inverse PCR (IPCR) described by Ochman et al in 1988 is a method for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. The method uses the polymerase chain react ...

Inverse PCR for PAC-end sequencing from Brad Barbazuk Goal is to generate PCR fragments that contain the ends of PAC inserts that can be sequenced. For inverse PCR we cut the PAC once in the vector ( ...

IntroductionThe diversity of pathogenesis presents a spectrum of challenges to the researcher who is required to utilize a wide variety of tools and techniques including those of a histological immun ...