Description
Formerly DNA Extraction Control 560.
DNA Extraction Control not only enables users of a diagnostic qPCR assay to determine if there are inhibitors in the PCR assay, but also to validate the success of the extraction step, reducing the chance of obtaining a false negative result in the sample DNA.Product Highlights
- Simple - streamlined protocol for straightforward validation of DNA extraction and determination of qPCR assay inhibition
- Sensitive - control assay identifies even small effects on DNA extraction and inhibition of amplification
- Optimized - control DNA has a sequence with no known homology to any organism thereby avoiding detection of sample DNA
- Specific - probe-based assay designed specifically for real-time PCR assays
- Flexible - ideal for use with a wide range of sample types, including inhibitor-rich samples like blood, urine and sputum samples
Product Description
A common practice in qPCR is to add a known amount of spiked control DNA after DNA extraction, this monitors PCR inhibition but has no value as an extraction control. The ideal situation is to have the test sample and internal control undergo the same processing prior to qPCR. Meridian has developed the qPCR Extraction Control, which more closely mimics the test sample, as compared to spike controls. Genetic material from the test sample and the qPCR Extraction Control is simultaneously extracted by common extraction methods, with the extraction control being as sensitive to inhibition and extraction failure as the test sample.
The qPCR Extraction Control cells are of a known concentration, containing the Internal Control DNA sequence. This sequence contains no known homology to any organism and, importantly, has minimal interference with detection of sample DNA. The qPCR Extraction Control cells are spiked into the lysis buffer with the target sample, prior to DNA extraction. Control Mix, which includes primers and probe, is then added to the reaction mix before amplification. Signal derived from the Internal Control DNA confirms the success of the extraction step. qPCR Extraction Control also monitors co-purification of PCR inhibitors that may cause biased or false amplification patterns.
Applications
- Pathogen detection
- Cancer risk assessment
- Gene expression analysis
- Copy number variation (CNV) analysis
- Genotyping
- Viral loading
Real-Time PCR Selection Chart
Application Note
qPCR Extraction Control Orangeebiomall.com
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转录的只是插入的片断,怎么会跟模板一样大呢,一般情况下应该比模板小多了。我都是跑电泳,严格来说应该跑变性的;嫌麻烦可以非变性,快速(防止RNA降解)十分钟即可;抛出来的带会有两条,上面大的是模板,下面一条比较亮的是RNA,那么你的转录就 成功了。
PCR第一个步骤如果没记错的话应该叫“变性”, PCR的话是不需要解旋酶的,直接利用DNA的高温变性特性,在高温下,使氢键断开,DNA双链恢复为单链。 (还有解旋酶好像是解除双螺旋的结构,是一种拓扑异构酶,对于氢键的打开没有贡献吧
2.模板浓度测定:分光光度计或NanoDrop
3.逆转录:逆转录试剂盒(或者一步法试剂盒),这一步可以用普通PCR做,也可以用水域做。
4.荧光定量PCR试剂:通常有用SYBRGreenMix做的,但是这里建议你用EvaGreen做,灵敏度和平行性都要好于SYBRGreen,并且如果你那是ABI或者Stratagene的PCR如果用SYBRGreen还需要加一步Rox很麻烦。
5.其他:除了以上的那些还需要离心管、PCR管或板(Axygen反应比较好)、移液枪等,暂时就想到这么多。
对于新手来说购买反转录试剂盒是比较理想的,买一个kit看看说明书操作就可以了。推荐两款kit TAKARA和HaiGene,这两款试剂盒都能对RNA样品中的gDNA有效去除,因此对RNA质量的要求不高。TAKARA的RR047A操作方便,反转录温度为37℃,对于大多数试验来讲是满足要求的。HaiGene的D0401操作要多一个步骤,但其反转录温度是55℃(耐高温的反转录酶),提高了反转录温度使得高GC含量、复杂模板、长mRNA的模板都能有效反转录,因此其反转录效率更高,更能够获得样本中基因的真实表达量。如果后续试验是RealTime PCR、ORF克隆、高GC含量、或者你的待研究基因结构复杂程度未知,还是选用耐高温的反转录酶更理想。对于反转录高手来说,直接购买反转录酶、再购买Rnase Inhibitor自己配制反转录体系就可以了。对于样本量大的课题组来讲,相对还是比较经济的。选择反转录酶时仅需要考虑是否需要耐高温的酶,来克服目的基因的复杂结构就可以了。PROMEGA、TAKARA、HaiGene、 TRANSGEN等品牌的反转录酶性价比还都是不错的。Life、NEB的也不错,不过性价比一般,不一一解释了。
首先,不否认它比大多数国产的试剂盒做得好,提取的纯度、方便度和量,都还很不错。
其次,它是日本的。so我觉得实验室用用天根的,就非常好了。拒绝日货,从实验室做起。