PCR技术

聚合酶链式反应（PCR） 聚合酶链式反应（Polymerase Chain Reaction，PCR）是体外酶促合成特异DNA片段的一种方法，为最常用的分子生物学技术之一。典型的PCR由（1）高温变性模板；（2）引物与模板退火；（3）引物沿模板延伸三步反应组成一个循环，通过多次循环反应，使目的DNA得以迅速扩增。其主要步骤是：将待扩增的模板DNA置高温下（通常为93℃-94℃）使其变性解成单链；人工合成的两个寡核苷酸引物在其合适的复 ...

问 题可能原因建议解决方法RT-PCR灵敏度：在琼脂糖凝胶分析中看到少量或没有RT-PCR产物RNA被降解在用来验证完整性之前先在变性胶上分析RNA使用良好的无污染技术分离RNA在将组织从动物体取出后立刻处理在100％甲酰胺中储存RNA如果使用胎盘RNase抑制剂，不要加热超过45℃或pH超过8.0，否则抑制剂或释放所有结合的RNase。而且，在≥0.8mM DTT时加入RNase抑制剂，一定要存在DTT。RNA中包含 ...

Single Primer ("Semi-Random") PCRJuly 26, 2000 ECKDescriptionSingle primer PCR allows amplification from known to unknown regions in chromosomes, phage, plasmids, large PCR products and other sources of DNA.At sufficiently low stringency, any primer will misprime while continu ...

The following colony PCR protocol has been designed to be performed in individual reaction tubes. We usually test three colonies from each transformation along with a wild-type control. A series of five different PCR tests are performed on each colony to confirm both of the novel recombination ...

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聚合酶链反应一单链构象多态性分析 (PCR-SSCP) 　 聚合酶链反应-单链构象多态性分析（Single Strand Conformation Polymorphism Analysis of Polymerase Chain Reaction Products, PCR-SSCP）是近年来发展起来的一种基因分析方法。 　PCR-SSCP分析的基本程序为：首先PCR扩增特定靶序列，然后将扩增产物变性为单链，进行非变性聚丙烯酰胺凝胶电泳。在不含变性剂的中性聚丙烯酰 ...

（一）、仪器设备 英国Thermo Hybaid原位PCR仪。 （二）、操作流程 1、原位PCR步骤 1）预处理：（1）切片常规脱蜡；（2）0.2mol/L HCl处理10min；（3）5μg/ml蛋白酶K消化组织37℃10min；（4）Nase消化组织37℃ 30min；（5）梯度酒精脱水，室温干燥。2）原位扩增：（1）切片滴加特异性序列引物30μLPCR扩增反应液，覆盖硅化盖玻片，石蜡油封边；（2）PCR热循环：94℃，1min；55℃，1min；72℃，1.5min ...

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 (NB: higher concn for total genomic DNA; lower for plasmid / purified DNA / virus DNA target) Buffer: use proprietary or home-made 10x rxn m ...

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Polymerase Chain Reaction (PCR) cont.Choice of Polymerases for PCROne of the important advances which allowed development of PCR was the availability of thermostable polymerases. This allowed initially added enzyme to survive temperature cycles approaching 100 °C.Thermost ...

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General Guidelines for Long-PCR Conditions and Enzyme Mixtures==========Following the results of Cheng et al. (1) we have had success using Tth (ABI/Perkin-Elmer) as the main-component polymerase and Vent (New England Biolabs) as the fractional-component polymerase. For PCR with low-co ...

Long-PCR Reagents and Guidelines from George Church as Modified from Cheng et al. (1) General Guidelines for Long-PCR Conditions and Enzyme Mixtures==========Following the results of Cheng et al. (1) we have had success using Tth (ABI/Perkin-Elmer) as the main-component polymerase and Vent (New ...

General Guidelines for Long PCR Conditions and Enzyme MixturesEfficient Long PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Following the res ...

End repair: Add 5-10 units of T4 DNApol and incubate at 37C for 5 minutes. Make solution to 2.5 M NH4OAc and add 1 volume of 95% ethanol; let sit for 5 minutes at RT and spin at 14K x g for 20 minutes. Wash with 70 % ethanol, dry and resuspend in 20 ul T4 PNK, 20 pmol ATP and 5-10 units T4 PNK. Incubate at 37 C for 1 hour. Ligation reaction: Blunt end liga ...

When designing PCR experiments in which the synthesized DNA fragment is to be subsequently digested with a RE, it is very important to determine how many extra nucleotides should be added to the 5’-end of the PCR primer next to the introduced recognition site to ensure efficient cleavage with the app ...

Place the sephadex measuring plate (MultiScreen ?Column Loader) on a clean piece of saran wrap. Pour some sephadex onto the plate (takes about 3.3 g). Scrape the surface with the Multiscreen metal plate with the plastic scraper so that the sephadex will fill all 96 wells. Place Polyfiltronics 96-we ...

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 suboptimal and over-digestion 3.1.4. DNase digestion 3.1.5. Direct incorporation of the rep ...

Cloning PCR products using TA vectorsby Paul N. Hengen, Ph.D. *Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column compares some of the commercially availa ...

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