PCR技术原理

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 revolutionized the detection of DNA and RNA. As little as a single copy of a particular sequence can ...

PCR基础 聚合酶链式反应（PCR）过程利用模板变性，引物退火和引物延长的多个循环来扩增DNA序列。这是一个指数增长的过程，因为上一轮的扩增产物又作为下一轮扩增的模板，使其成为检测核酸的一种非常灵敏的技术。一般，经过20-30个循环得到的扩增产物就足够在溴化乙锭染色的凝胶上观察到。反应包括几个成分（表1）。模板可以为纯化的基因组或质粒DNA；由RNA转化得到的cDNA；或未经纯化的粗制生物样品，如细菌克隆或噬菌斑。引 ...

Hi everyone, I'll start doing rt-pcr using sybr green chemistry and already ahve the primersPrimer design tips for real time suggest Tm to be 58-60 or at least over 50.Why is that? How important is that? My Tm's are all around 53 and I guess that in any case I can set the temperature in the annealing step of the PCR to fit my prim ...

I have been working with the Real-time PCR and have run into a brick wall. My standard curve is running that all samples no matter what the dilution factor are amplifying at the same Ct. I am also getting amplification at that same Ct from a negative control (no template). Our first reaction was contamination, b ...

hi, I am going to do real-time PCR using Smart Cycler, but I have never done this experiment, I do not know how to design primers,such as the length of primers,the size of PCR product and the annealing temperature et al. If you happen to know the knowledge about it, would you like to sharing with me? Thanks a lot! -geness- He ...

The gene we are interested in looking at is not very abundant. At 100 ng/ul, the CT value is 30.9In the literature that I have looked at, people seems to using much lower levels of cDNA. I would actually like to increase the concentration of cDNA to 200 ng/ul but am wondering if this can cause any problems.I use SYBR green ...

1 原理 　　RT—PCR是一种将cDNA合成与PCR技术结合分析基因表达的快速灵敏的方法，主要用于对表达信息进行检测或定量分析,还可以用来检测基因表达差异而不必构建cDNA文库克隆cDNA。 RT-PCR的模板可以为总RNA或poly(A) 选择性RNA。逆转录反应可以使用逆转录酶，以随机引物、oligo(dT)或基因特异性的引物（GSP）起始。在实际应用中，RT—PCR又常常分为一步法RT—PCR和两步法RT—PCR。 2 实验流程3 特点　　一步法 ...

hi evry body!any body known realtime sybergreen concentration in pcr reaction.i planed external addition all sybergreen kit (roche) kontent??? -sef- the only real advice i can give you is to not add sybr green externally, but to buy it in a kit - like Sigma SybrGReen jumstart ready mix which is what i use (lots of ...

Hello everyone. My day was ruined by these two peaks~~~ It's supposed to be one, which represents single product after amplification. Does any one know the reason? I've checked the sequences of both primers. Blast gives single targeted gene sequence. -meflower- QUOTE(meflower @ Jul 11 2006, 08:33 PM) 59 ...

Hi, I am wondering about the choice of housekeeping genes for my assay. If the gene of interest is a nuclear gene, is it better/ worse to use a mitochondrial gene as a housekeeping one. Are there any major pros/cons of the most popular housekeeping genes, like 18 S rRNA or GAPDH?thanks -smurray- I use beta-actinfor ...

I have a quick question. In our lab we have had a great deal of sucess using qRT RT-PCR on the lightcycler. We are currently trying to examine gene expression in exfoliated epithelial cells, however the cell numbers are such that we only get a very small amount of RNA

Hi all, I am doing some QPCR experiments with SYBR Green using cDNA and my primers never seem to work properly. I re-designed it 3 times already, the first time the primers looked promising- from the melting curve there were no primer dimers but then the CT values were flutuating up and down for my dilutions. So I re- ...

Greetings,In my Real-Time qRT-PCR experiments, I employ the standard curve method for quantification of gene expression. However, standard curves seem to be a huge hit-or-miss procedure for me, even with genes that are well-established to work well with Real-Time such as GAPDH.At times, I am able ...

Hi all,I have a dilemma - I've run a dilution series on my housekeeping gene and gene of interest to test for equal primer efficiencies, graphing Delta-Ct vs. log concentration, and my primers pass the test, with a slope much less than.1. However, when I graph the standard curves for each primer pair, the slopes a ...

Hi, everyone, I am newcomer and have started Real Time PCR for 2 months. After all, I have designed the primers sets again using Primer Express 1.0 and using Oligo-4.0 to check if there are suspected dimer formation. However, most of primers designed by Primer Express seem to have many dimer formation by upper ...

hi again,still getting a signal in my template free negative control (nuclease free water instead of template)have changed supply of nuclease free water and purchased new primers and still get a signal?????what is going wrong!?!?!?!?!?!?! -flashboy- The new primers were made up in the new water right?D ...

主要内容RT-PCR原理RT-PCR步骤常见问题分析两步法RT-PCR一步法RT-PCR两步法与一步法RT-PCR比较RT-PCR主要用途分析基因的转录水平获取目的基因合成cDNA探针逆转录酶的选择AMV：禽成髓细胞瘤病毒，逆转录酶和RNA酶H活性。最适42℃，最新版本可达60 ℃ （Bioflux公司）。MMLV：Moloney 鼠白血病病毒，逆转录酶，RNA酶H活性较弱。最适37℃，最新版本42 ℃（赛百盛）Super RNaseH- R ...

hi there,i am noew constantly getting a late peak (30+ cycles) in the negative controls for my real-time pcr (SYBR Green, iCycler). i have even bought in new nuclease free water to run as my negative contro, and still get these peaks... does this mean my primers are contaminated???? -flashboy- Your working prim ...

RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection as ...

PCR引物设计原则 PCR引物设计的目的是为了找到一对合适的核苷酸片段，使其能有效地扩增模板DNA序列。因此，引物的优劣直接关系到PCR的特异性与成功与否。要设计引物首先要找到DNA序列的保守区。同时应预测将要扩增的片段单链是否形成二级结构。如这个区域单链能形成二级结构，就要避开它。如这一段不能形成二级结构，那就可以在这一区域设计引物。现在可以在这一保守区域里设计一对引物。一般引物长度为15~30碱基，扩增片段长 ...