I'm having some difficult trying to do a 5' and 3' RACE protocol and I was wondering if anyone might be able to help. I'm using a Epicentre ExactSTART kit.
The first thing I am wondering if whether I need to "clean up" my RNA sample. Previously, I isolated total RNA from cells using RNA-STAT 60. The first step of the RACE protocol is treating with alkaline phosphatase to remove the 5' phosphate group from 5' mono-,di-,tri-phosphorylated RNA in the total RNA sample (while not affecting RNA containing the 5'-cap structure). Then my protocol says I need to clean up the sample by phenol-chloroform extraction. The problem I am having is that I can't see the pellet after this clean up step (it could be invisible) and I'm afraid I'm losing RNA in this process. I was just wondering if I can skip this step entirely and proceed with the rest of the protocol. The downside is that I won't remove the phosphate groups.
5' and 3' RACE Help
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