q**********0
发帖数: 335 | 1 Recently, I tried twice by using MEGAscript kit-making RNA Probes (32P-label
GTP and incorporated) in sourthern Blot. However, all failed. The probe is
single-stranded RNA, it should blot the DNA template. I don't know what's
wrong with it. Anybody have the experience on this? Please help. Thanks. |
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q**********0
发帖数: 335 | 2 Recenly I use T3 megascript do in vitro transcription. The results is always
bad, the OD260/280 is about 1.53. A big white pellet can be seen after
isopropanol precipitation and RNA concentratin measure is about 1.0 ug/ul.
However, when run a RNA gel, the band is very faint even using 5 uL. I don't
know why? Maybe there is protein contamination? or RNAse digestion? Please
help. Thanks a lot! |
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f****n
发帖数: 114 | 3 小弟最近在做RNA in vitro transcription，用的是ambion 的MEGAscript kit。
可是不论是用PCR产物直接做模板，还是连接到质粒上再线性化做模板，都得不到sharp
的条带，size比预期小很多并且有拖尾，但kit里阳性对照可以出来。
已经排除了RNase污染。估计是DNA模板纯度的问题，请问各位PCR产物用啥方法纯化？
另外，我在T7promoter序列前面加了7个碱基，难道是这个影响酶的效率？
请各位高手帮忙！ |
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