Rapid amplification of cDNA ends (RACE) is a technique used to obtain the full-length sequences of RNA transcripts. RACE can provide the sequence of an RNA transcript from a short, known sequence within the transcript all the way to the 5" end (5" RACE-PCR) or 3" end (3" RACE-PCR) of the RNA.
The SMARTer RACE 5"/3" Kit is an improved version of the SMARTer RACE cDNA Amplification Kit. The SMARTer RACE 5"/3" Kit provides improved sensitivity, less background and higher specificity over the previous generation of kits. First-strand cDNA generated with this kit can be used directly in 5"- and 3"-RACE PCR reactions. The SMARTer protocol does not involve any of the adaptor ligation steps that other RACE kits incorporate, making the protocol shorter and significantly easier to execute. The new kit is also designed to accommodate larger RNA input volumes (up to 11 µl) and has been shown to perform more efficiently on challenging targets (e.g., those that are long, GC-rich, etc.).
Rapid amplification of cDNA ends (RACE) is a technique used to obtain the full-length sequences of RNA transcripts. RACE can provide the sequence of an RNA transcript from a short, known sequence within the transcript all the way to the 5" end (5" RACE-PCR) or 3" end (3" RACE-PCR) of the RNA.
The SMARTer RACE 5"/3" Kit is an improved version of the SMARTer RACE cDNA Amplification Kit. The SMARTer RACE 5"/3" Kit provides improved sensitivity, less background and higher specificity over the previous generation of kits. First-strand cDNA generated with this kit can be used directly in 5"- and 3"-RACE PCR reactions. The SMARTer protocol does not involve any of the adaptor ligation steps that other RACE kits incorporate, making the protocol shorter and significantly easier to execute. The new kit is also designed to accommodate larger RNA input volumes (up to 11 µl) and has been shown to perform more efficiently on challenging targets (e.g., those that are long, GC-rich, etc.). The kit includes our highly robust SeqAmp DNA Polymerase for amplification of products, and the In-Fusion HD Cloning Kit for efficient cloning of RACE fragments in the provided linearized vector. The SMARTer RACE 5"/3" procedure combines SMART first-strand cDNA synthesis technology with a powerful suppression PCR protocol that vastly reduces the background amplification commonly associated with RACE protocols.
- Easy to use—the SMARTer RACE protocol is performed in a single tube, and minimizes handling of both your RNA sample and your synthesized cDNA. Total hands-on time is only four hours.
- Complete kit—the SMARTer RACE 5’/3’ Kit contains all the necessary components (save gene-specific primers) to streamline your process. With this single kit, you can begin first-strand cDNA synthesis and proceed through cloning RACE fragments, recovering successful clones on day two.
- Requires only 10 ng of total RNA—our SMARTer RACE method allows you to utilize small samples, including biopsies, tissue dissections, needle aspirations, and embryonic and rare disease tissues. This optimized protocol significantly reduces non-specific background, which is helpful for analysis of small inputs.
- Specific enrichment for 5" ends—our carefully designed, chemically treated SMARTer Oligo preferentially hybridizes to the 5" ends of the cDNA being synthesized. Using this SMARTer Oligo, our procedure enriches cDNA pools for 5" sequences, thus increasing the likelihood you will amplify the entire sequence of your gene or the upstream regulatory regions.
- No RNA pretreatment required—our SMARTer RACE method requires no RNA pretreatment with DNase. This protocol works with total RNA, as well as with samples that may be contaminated with genomic DNA.
Related products
Marathon cDNA Amplification Kit
The Marathon cDNA Amplification Kit method employs a specially designed adaptor that significantly reduces background and permits both 5"- and 3"-RACE reactions ((Bertling, Beier, and Reichenberger 1993; Frohman 1993) to be performed using the same template. Marathon cDNA amplification can be used to quickly characterize multiple RNAs identified by expressed sequence tags (ESTs), differential display, RNA fingerprinting, or cDNA subtraction. Marathon cDNA synthesis begins with poly A+ RNA and a modified lock-docking oligo(dT) primer that contains two degenerate nucleotides at the 3" end. These nucleotides position the primer at the beginning of the poly A tail, eliminating the 3" heterogeneity inherent with conventional oligo(dT) priming. Following cDNA synthesis, blunt ends are created and the Marathon Adaptor is ligated to both ends of the double-stranded cDNA.
NOTE: The Marathon cDNA Amplification Kit requires the Advantage 2 PCR Kit (Cat. # 639206).
RACE-ready cDNAs
We also offer RACE-ready cDNAs, which are double stranded cDNAs made from high-quality premium poly A+ RNA and ligated to the Marathon Adaptor. These cDNAs are ready for 5"- and 3"-RACE PCR and are available from a wide range of tissues and cell types.
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1、直接竞争，标记抗原，与检测样品中的抗原竞争抗体。
2、间接竞争，标记抗体，固相抗原与液相抗原（样品）竞争标记抗体。
3、定义
间接竞争法的模型：包被抗原，用HRP-抗体与样本一起加入。样本中的Ag与Solid-Ag竞争HRP-Ab，固相吸附的HRP-Ab与样本中的Ag浓度成反比。
直接竞争法的模型：包被抗体，用HRP-抗原与样本一起加入。样本中的Ag与HRP-Ag竞争Solid-Ab，固相吸附的HRP-Ag与样本中的Ag浓度成反比。
4、竞争法的理论基础：是限量抗体。只有在限量抗体基础上，两种抗原才会形成竞争关系。
5、、间接竞争法具备较高灵敏度原因。
直接竞争法里，标记抗原与待测抗原均是液相，与抗体的结合机会是一样的，例如有1份标记抗原与1份待测抗原竞争1份抗体，那么有50%的标记抗原能与抗体结合，所以标记抗原的相对结合率为50%。间接法里，固相抗原与抗体的接触面积较小，固相抗原与待测抗原的结合抗体机会是不平等的，接近顺序饱和法，即只有与待测抗原结合剩余的抗体才会与固相抗原结合，同样有1份固相抗原与1份待测抗原竞争1份抗体时，基本上抗体会被待测抗原中和掉，与固相抗原结合的抗体非常少。固相抗原的相对结合率为0%。因此，间接法的抑制曲线斜率会大于竞争法。
因为抑制率越大则斜率越大，从而灵敏度越大。（假设零管变异5%，以两倍SD为灵敏度限，则为90%相对结合率，则间接法可以在较低的待测抗原浓度达到这一相对结合率，因此灵敏度要高。）
在进行系统放大时，间接法一般可以使用酶标二抗。因为二抗可以针对抗体的多个部位，所以存在放大效应，从而能提高间接法的灵敏度。直接法一般难以进行放大，常用的有生物 素化抗原与酶标亲和素，但模式上似乎不存在放大效应。
6、间接法的高灵敏度难以实现的原因：
双抗体 夹心的免放（IRMA）模式刚出现时，也被模型证明灵敏度优于竞争法的放免（RIA），原因也是较大的斜率，但是IRMA的高灵敏度一直到单抗发展后才得以实现。
GM实验：试剂说明书上要求20分钟显色，10分钟的时候看一下，差不多就可以加终止液，但是我的标曲显色特别快，2分钟最低浓度蓝色就就深了，此时样本颜色还很浅很浅，我只能一块儿加终止液。所以，标曲显色过快，可能是什么原因？
（ps:第一列是标曲，后面是样本）
