Plasmid Info:
Plasmid Information
Product Name: pSF-pA-PromMCS-FrCFP
Product Code: OG615
Size (bp): 4613 bp
Bacterial Antibiotic Selection: AmpR
Origin and Compatibility: pUC high copy derived from pBR322
Bacterial Copy Number: 500-700 per cell
Promoter:
Plasmid Purpose:
Frosty CFP (Cyan Fluorescence Protein) expression plasmid with a multiple cloning site upstream to allow your preferred promoter to be inserted. Also contains a poly A site upstream to reduce background readthrough in mammalian systems
Sequence and Map:
Other Info:
Transcription Termination:This plasmid contains three alternative transcription terminators for mammalian bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.
Cloning:
Cloning in a gene:This plasmid contains a gene within the main multiple cloning site (NotI-ClaI). Any plasmid that we sell where the gene is this configuration will be located in the exact same position in relation to the start and stop codon of the gene. The only exceptions to this rule are fusions proteins where the fusion gene may be positioned at the front or end of the MCS to allow gene fusion.
By positioning all of our genes in the same location it allows them to be transferred between plasmids using the same cloning method and restriction sites regardless of the plasmid being used from our product range. Inserting a new gene into this plasmid should be easily possible using a range of standard restriction enzyme sites that flank the gene currently in the vector.
Multiple cloning site notes:In the multiple cloning site there are two important restriction sites called BsgI and BseRI sites. These sites both cut the DNA at the same position and cleave the stop codon of the gene in the multiple cloning site in this plasmid thereby producing a TA overhang. This overhang is compatible with any of our peptide or reporter fusion tag plasmids also cut with either of these enzymes. This allows seamless C-terminal fusions to be made with the gene in this multiple cloning site using a single cloning step from our C-terminal peptide and reporter tag product range. Normally the easiest method is to clone the C-terminal tag from our other plasmid products into this plasmid using BsgI or BseRI and the downstream ClaI restriction site.
BseRI and BsgI sites are non-palindromic and cleave a defined number of bases away from their binding sites. This allows them to cut the upstream stop codon in the gene in this plasmid regardless of the gene sequence.
IP Status:
Intellectual Property StatusThis product is part of our SnapFast plasmid range, for more information on the Intellectual property status of this plasmid please click here
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在这种测定方法中有3种必要的试剂：①固相的抗原或抗体（免疫吸附剂） ②酶标记的抗原或抗体（标记物）③酶作用的底物（显色剂）
测量时，抗原（抗体）先结合在固相载体上，但仍保留其免疫活性，然后加一种抗体（抗原）与酶结合成的偶联物（标记物），此偶联物仍保留其原免疫活性与酶活性，当偶联物与固相载体上的抗原（抗体）反应结合后，再加上酶的相应底物，即起催化水解或氧化还原反应而呈颜色。
其所生成的颜色深浅与欲测的抗原（抗体）含量成正比。 这种有色产物可用肉眼、光学显微镜、电子显微镜观察，也可以用分光光度计（酶标仪）加以测定。其方法简单，方便迅速，特异性强。向左转|向右转
ELISA双抗体夹心法(enzyme linked immunosorbent assay——sandwich technique)的原理是将特异性抗体结合到固相载体上形成固相抗体，然后和待检血清中的相应抗原结合形成免疫复合物，洗涤后再加酶标记抗体，与免疫复合物中抗原结合形成酶标抗体-抗原-固相抗体复合物，加底物显色，判断抗原含量。
生物帮有相关介绍。编码RNA http://doc.bio1000.com/show-3399.html
如果用双抗体夹心法，第二个抗体可以用多抗，这样保证你的抗原会被结合，多抗可以直接带HRP标记或者可以用HRP标记二抗去和多抗结合。
1.标准曲线的标准品是否一定要梯度稀释，为什么？我试过非梯度稀释的，也可以达到线性R2=0.99.
2.我用了CurveExpert做标曲，自动搜索后发现有10种提供的方程，各种形式的，其中一个十分适合我的实验结果（LogisticModel），而其他的感觉又不适合，因为结果常常为负值。这又是为啥捏？
3.实验的酶标仪最大OD值可以测到4，如果我的测量结果在1.3，是否像其他人所说的＞1了就不准确了。
4.利用夹心法进行定量分析是否一定要使用线性方程？
不好意思啊，一下问了这么多问题，最近做了一个月的ELISA，完全摸不清头脑啊。谢谢各位了
1、重复性不好；
2、收自身抗体、嗜异性抗体等干扰，易出现假阳性；
3、不论仪器和手工操作，干扰因素较多。影响最大的是温度和时间。1、直接法（direct ELISA）将抗原直接固定在固相载体上，参加酶符号的一级抗体，即可测定抗原总量，此一级抗体的特异性非常重要。优势：操作手续简略，因无须运用二抗可防止交互反响。缺陷：实验中的一抗都得用酶符号，但不是每种抗体都适合做符号，费用相对进步。2.间接法（indirect ELISA）此测定办法与直接法相似，不一样在于一级抗体没有酶符号，改用酶符号的二级抗体去辨识一级抗体来测定抗原量。优势：二抗能够加强信号，并且有多种挑选能做不一样的测定剖析。不加酶符号的一级抗体则能保存它最多的免疫反响性。缺陷：交互反响发作的机率较高。3.双抗体夹心法（sandwich ELISA）被检测的抗原包被在两个抗体之间，其间一个抗体将抗原固定于固相载体上，即捕捉抗体。另一个则是检测抗体，此抗体可用酶符号后直接测定抗原的量；或不符号，再透过酶符号的二级抗体来测定抗原的量。这两种抗体有必要当心选择，才可防止交互反响或竞争一样的抗原联系部位。优势：高活络、高专一性，抗原无须事前纯化。缺陷：抗原必定得具有两个以上的抗体联系部位。4.竞争法（competitive ELISA）样本里的抗原（自在抗原）和纯化并固定在固相载体上的抗原（固定抗原）一同竞争一样的抗体，当样品里的自在抗原越多，就能够联系越多的抗体，而固定抗原就只能联系到较少的抗体，反之亦然。经清洁过程，洗去自在抗原和抗体的复合物，只留下固定抗原和抗体的复合物，拿来与只要固定抗原的对照组成果相比拟，依据呈色区别就可计算出样品里的抗原含量。优势：可适用比拟不纯的样本，并且数据再现性很高。缺陷：全体的敏感性和专一性都较差。本数据来源于百度地图，最终结果以百度地图最新数据为准。
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