Description
Product Highlights
- Fast – simple extraction of high purity total RNA from six samples, ideal for use in all applications, in as little as 30 minutes
- High-performance – recovery of consistently high quality RNA from a wide variety of animal, bacterial and plant cells and tissues and cell-free samples
- Efficient – optimized lysis conditions and column matrix for recovery of up to 70 mg of high purity RNA from each sample
- Convenient – includes all necessary components, including filters (shredders) and DNase I
- Safe - no hazardous phenol/chloroform extraction, CsCl centrifugation, LiCl or alcohol precipitation
Product Description
The ISOLATE II RNA Mini Kit provides a simple, efficient column-based method for the isolation of total RNA from a wide variety of starting materials, without the need for hazardous reagents such as phenol.
By combining the stringency of guanidinium-thiocyanate lysis with the speed and ease-of-use of silica-membrane purification, the ISOLATE II RNA Mini Kit provides a fast method for the purification of high-quality total RNA from animal and plant cells and tissues as well as cultured cells, bacterial cells, yeast, biological fluids and cell-free samples.
Biological samples which are sometimes difficult to process i.e. mouse tissue (liver, brain), various tumor cell lines, Streptococcus and Actinobacillus pleuropneumoniae, will yield high-quality RNA with the ISOLATE II RNA Mini Kit.
The online product manual has protocols for purifying total RNA from cultured cells, tissues, yeast, bacteria, biological liquids, paraffin embedded tissue and RNAlater® treated samples. There is also a protocol for a convenient on-column DNase treatment, using RNase-free DNase I that is supplied with the kit, for applications that are sensitive to very small amounts of DNA.
The ISOLATE II RNA Mini Kit has been designed to deliver optimal performance in RT-qPCR in conjunction with either the SensiFAST cDNA Synthesis Kit and SensiFAST Real-Time PCR Kits, or the SensiFAST One-Step Real-Time RT-PCR Kits. Additionally, the ISOLATE II RNA Mini Kit can be used to purify samples for PCR and RT-PCR amplification using the Tetro cDNA Synthesis Kit and any enzyme from the Meridian PCR portfolio, including MyTaq DNA Polymerase.
Applications
- RT-qPCR
- End-point RT-PCR
- Northern, dot and slot blotting
- Array analysis
- Poly A+ RNA selection
- RNA-Seq
Nucleic Acid Isolation Guide
Download the ISOLATE II Guide with detailed product descriptions and performance data to help you choose the best product for your researchISOLATE II Selection Chart
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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。