EmeraldAmp GT PCR Master Mix is a loading-dye-added version of EmeraldAmp MAX PCR Master Mix that is optimized for great performance and conveniencein both standard and high-throughput PCR applications. This master mix includes an optimized buffer, PCR enzyme, dNTP mixture, gel loading dye (green), and a density reagent in a 2X premix format. Simply add primers and DNA template. Following PCR, amplicons can be used directly in several ways.
EmeraldAmp GT PCR Master Mix is a loading-dye-added version of EmeraldAmp MAX PCR Master Mix that is optimized for great performance and conveniencein both standard and high-throughput PCR applications. This master mix includes an optimized buffer, PCR enzyme, dNTP mixture, gel loading dye (green), and a density reagent in a 2X premix format. Simply add primers and DNA template. Following PCR, amplicons can be used directly in several ways.
PCR tube contents can be loaded directly onto an agarose gel for electrophoresis or used directly in downstream applications such as restriction enzyme digestion, TA cloning, and direct sequencing. EmeraldAmp GT PCR Master Mix can be used to amplify genomic targets up to ~5 kb and is compatible with GC- and AT-rich targets.
When 5 µl of the loading-dye-added PCR master mix is used for electrophoresis on an 1% Agarose L03 gel, the blue dye front is detected around 3–5 kb, and the yellow dye is detected below 50 bp. These dyes have absorptions at approximately 260 nm and 420 nm, respectively. The dyes can be removed by gel purification or PCR cleanup using the NucleoSpin Gel and PCR Clean-Up kit (where available), if necessary.
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如果要测量细胞表面2个受体的比率,最好用流式细胞仪来测量
跑到还有半小时的时候,跳出一个对话框:“analysiscannotproceed:notenoughsamplesdefined”,最后做出来的结果有扩增曲线但是没有熔解曲线,请各位大神帮忙解释一下。
荧光定量PCR较普通PCR不同的一点就是可以实时检测PCR扩增产物,从而可进行绝对定量或者相对定量。
半定量反转录-聚合酶链反应(semi-quantitative reverse transcription and polymerase Chain reaction ,SqRT-PCR)是近年来常用的一种简捷、特异的定量RNA测定方法,通过mRNA反转录成cDNA,再进行PCR扩增,并测定PCR产物的数量,可以推测样品中特异mRNA的相对数量。
定量RT-PCR(quantitative reverse transcription and polymerase Chain reaction ,qRT-PCR)是在用一步法或两步法,在PCR反应体系中加入荧光基团,利用荧光信号积累实时监测整个PCR进程,最后通过标准曲线对未知模板进行定量分析的方法。
半定量RT-PCR需要跑电泳,根据条带亮度的强弱来判断模板拷贝数的高低或者是表达量的高低,而定量RT-PCR则无需电泳可以实时监测整个PCR的全程并且由给出的Ct值及Standard Curve来判断gene拷贝数的高低。
PCR(聚合酶链式反应)是利用DNA在体外摄氏95°高温时变性会变成单链,低温(经常是60°C左右)时引物与单链按碱基互补配对的原则结合,再调温度至DNA聚合酶最适反应温度(72°C左右),DNA聚合酶沿着磷酸到五碳糖(5'-3')的方向合成互补链。
恒温PCR和实时荧光定量PCR的不同,是不同在实时荧光定量PCR的系统中加入了荧光染料(SYBR Green 或Taqman 探针等等)。以SYBR Green为例,这种染料可以结合在双链的DNA上,当PCR不断进行时,每一次退火生成的双链DNA也在增加,荧光染料结合也越多,荧光也越强。在机器中有一个探测荧光的探头,可以定量检测荧光的强度,转换成数值。这样就可以实时记录反映体系中DNA的反应情况。
荧光PCR更有优势,因为荧光PCR灵敏度高于恒温PCR,同样价格也高一些。
还需要反转录用的引物、定量PCR用的一对引物、荧光信号源(荧光染料或荧光标记的探针,看你用什么策略来做定量PCR)
报价要看你需要服务商提供什么服务,如果你只提供实验材料,要求服务商做以上全部工作,只检测一个指标(一个基因的表达水平变化),价格大约每个样品350-500元,多一个指标大约多90元。
一般至少做一组数据两个样品嘛,所以你按1000块一组样品预算吧。
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