
SP6 RNA Polymerase is a DNA-dependent RNA polymerase that exhibits high specificity for the bacteriophage SP6 promoter sequence 5"-TAATCCACTGTGATAT-3". The enzyme can incorporate labeled or unlabeled ribonucleoside triphosphates into an RNA transcript. Large quantities of RNA can be synthesized from a DNA sequence cloned downstreamof the SP6 promoter due to the high processivity of SP6 RNA Polymerase. The enzyme is supplied in a buffer consisting of 10 mM potassium phosphate (pH 7.9), 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol.
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实验非常不顺,想构建CDNA文库,但是从mRNA开始屡次失败,考虑主要是纯化过程中损失过多。想从总RNA入手,但是不知道实验步骤。不知那位大侠能提供总RNA建库的实验步骤。另外我现在手头有OligoDT,RT酶,苦于没有第二连合成试剂盒,不知道能否用普通PCR试剂盒Teq酶替代第二链合成过程中的DNA聚合酶I。好像有种方法合成第二链时,不需要另外的引物,利用降解的RNA作引物即可,我想直接设定PCR两个循环,合成第二链,不知方法可行否?愁啊,等着毕业,时间紧急,恳请帮忙。谢谢。

