annealingtempfordegeneratedprimerPCRproblem
PCR primers should be free of significant complementarily at their 3'-termini as this favours formation of primer-dimer which reduce product yield. Formation of primer-dimer artifacts may also cause more serious problems, such as non-specific DNA synthesis (asymmetric PCR). (Help from the internet: http://www.basic.nwu.edu/biotools/oligocal...ml)Magnesium chloride is one of the main variables. It affect......阅读全文
LongPCR－Reagents－and－Guidelines
Long-PCR Reagents and Guidelinesfrom George Church as Modified from Cheng et al. (1)General Guidelines for Long-PCR Conditions and Enzyme Mixtures====
PCR实验指导与常见问题分析4
Fig. 25. Multiplex PCR of mixtures A-D comparing PCR programs with 2 (green) and 1 (yellow) minute extension time at 54° C annealing temperature. Comp
Taq酶PCR实验方法介绍
General AdvicePCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this tec
PfuDNA聚合酶PCR实验方法介绍
General AdvicePCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this tec
Cycle－Sequence－Reactions－For－Large－Insert－Plasmid－Templates
The following dye-labeled terminator reaction chemistries have been designed to balance conservation of reagents with the resulting sequence product s
PCR实验指导与常见问题分析5
MgCl2 concentrationRelationship between MgCl2 and dNTP concentrationdNTP concentrations of about 200µM each are usually recommended for the Taq polyme
Streptomyces:Protocols/PCR
Description Polymerase Chain Reaction (PCR) is a method of amplifying a specific DNA target sequence. The cycle involves denaturing the template doubl
PCR实验指导与常见问题分析7
11. All products in my multiplex reaction are weak. How can I improve the yield?Decrease annealing time in small steps (2º C)Decrease extension temper
引物设计原则（Principle－of－realtime－quantiation－PCR－primer－）
1、引物的长度一般为15-30bp，常用的是18-27bp，但不应大于38，因为过长会导致其延伸温度大于74°C，不适于Taq DNA聚合酶进行反应。2、引物序列在模板内应当没有相似性较高，尤其是3’端相似性较高的序列，否则容易导致错配。引物3’端出现3个以上的连续碱基，如GGG或CCC，也会使错误
3RACE－PCR
实验概要 This technique is used to obtain the 3'end of a cDNA, it requires some sequence information internal to the mRNA under study. The
Protocol－for－competitive－RTPCR
For quantifying mRNA, we use a competitive RT-PCR protocol with internal standard RNAs. These are added in a defined quantity to the RNA sample prior
Degenerate－PCR,－a－short－guide.
What is degenerate PCR? Degenerate PCR is in most respect identical to ordinary PCR, but with one major difference. Instead of using specific PCR pr
Standard－PCR－reaction
Steps for Standard PCR ReactionDesign primers. In general, primers should have the following properties:Tip: Primer3 is an excellent resource for choo
Multicolour－3DFISH－in－vertebrate－cells1
IntroductionMulticolour 3D-FISH in combination with confocal microscopy, 3D image reconstruction and quantitative image analysis is an efficient tool
primer－primer5怎么设计引物
首先打开软件左上角来操作file——new——dna sequence这一项可以把你复制的序列粘贴进去当然，你也可以选择另一个file——open——dna sequence去open一个新的文件。在接下来的界面里复制你的序列，于是就将序列导入了点击search进入下图界面开始设计引物在此图中有6个
DNA甲基化分析
The influence of methylation on the promoter activity and gene expression and the involvement of DNA methylation in carcinogenesis caused an extensive
Colony－PCR
Colony PCR is useful in determining whether or not a specific colony on a plate has a sequence you desire. Primers for the specific sequence should be
果蝇RNAi的实验中双链短RNA的合成（dsRNA)方法
实验概要We routinely produce dsRNA by in vitro transcription of a PCR generated DNA template containing the T7 promoter sequence on both ends (I. Prim
Experimental－Protocol－for－cDNA－Library－Construction
Experimental Protocol for cDNA Library ConstructionIdentify appropriate celltype over-expressing corresponding gene.Find out if transcription can be s
How－do－you－synthesize－your－dsRNA
We routinely produce dsRNA by in vitro transcription of a PCR generated DNA template containing the T7 promoter sequence on both ends (I. Primer Desig
其它PCR方法
· Standard PCR Protocol (Molecular Biology Techniques Manual)The followings are described in detailRecommended Reagent ConcentrationsRecommend
PCR经验总结（二）
5. touchdown PCR 原理很简单,但的确是一个很有用的方法。举个例子就OK, ANNEALING TEMP. 55度94 5min 94 30s 60 30s 72 1min 2cycles 94 30s 59 30s 72 1min 2cycles 94
PCR实验技巧2
5. touchdown PCR 原理很简单,但的确是一个很有用的方法。举个例子，ANNEALING TEMP. 55度 94 5min 94 30s 60 30s 72 1min 2cycles 94 30s 59 30s 72 1min 2cycles 94 30s 58 30s 72 1min
pcr实用技巧
增加PCR的特异性：1. primers design这是最重要的一步。理想的，只同目的序列两侧的单一序列而非其他序列退火的引物要符合下面的 一些条件a. 足够长,18-24bp,以保证特异性.当然不是说越长越好,太长的引物同样会降低特异性,并且降低产量b. GC% 40%~~~~60% c. 5&
整理的PCR资料.供大家分享.
PCR实验技巧增加PCR的特异性：1. prime理想的，只同目的序列两侧的单一序列而非其他序列退火的引物要符合下面的 一些条件a. 足够长,18-24bp,以保证特异性.当然不是说越长越好,太长的引物同样会降低特异性,并且降低产量b. GC% 40%~~~~60%c. 5'端和中间序列要多
果蝇RNAi的实验中双链短RNA的合成（dsRNA)方法
本文来自于哈佛大学医学院果蝇RNAi筛选中心的经典实验方法，专门用于果蝇RNAi实验方法。感谢哈佛大学医学院果蝇RNAi筛选中心的支持！Primer Designed dsRNATemplate SelectionPCRIn vitro RNA TranscriptiondsRNA Purifica
PCR－protocol
PCR reactionProtocol for 50µl reaction - adjust amounts if necessary, for a 20µl reaction use the same volumes of primer and dNTP-mix, but adjust the
Cloning－PCR－products－using－TA－vectors
Cloning PCR products using TA vectorsby Paul N. Hengen, Ph.D. *Methods and reagents is a unique monthly column that highlights current discussions in
Mouse－p27－PCR－Using－Gitschier－Buffer
Mutant allele: Neo-1 primer (CCTTCTATCGCCTTCTTG), plus mgK-3 primer (TGGAACCCTGTGCCATCTCTAT) produce a 0.5kB PCR product.Wildtype allele: mgK-3 (above
Degenerate－PCR
Degenerate PCR is in most respects identical to ordinary PCR, but with one major difference. Instead of using specific PCR primers with a given sequen