5' and 3' RACE Help - (Jun/03/2009 )
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.
You need to remove the phosphates so that the 5' end won't be filled while the 3' RACE is going on. If you leave the phosphatase in there it will de-phosphorylate the rNTPs meaning your reaction won't work.