I am using a T7 Megascript RNA kit from Invitrogen. I transcribe overnight at 37˚C, treat with DNase, phenol/chloroform extract, precipitate with isopropanol, then denature 30mins at 70˚C and let it slowly cool to RT to renature. In my final product there is a small white precipitate on the bottom when the tube should be clear. Is the dsRNA still usable / has anyone else experienced this? The RNA band was not entirely clean (faint larger band) when run on a gel.
Dear Cowan, Did you check concentration of extracted RNA by nanodrop?? What was concentration and 260/280 value??
Dear Quinn
From my experience, I am doing in vitro transcription manually without any kits. The white precipitate shouldn't be appeared until you proceed to RNA precipitation step. For me, I use 4 M LiCl to precipitate my product. Afterward, I check my dsRNA quality by non-denaturing electrophoresis. I mix my product with RNA loading dye 1:1 ratio. The band should be appeared as the big 2 strips around expected size of your product.
Bests,
I use the same kit for invitro transcription. But why do you process with phenol chloroform after DNase? I precipitate my RNA with lithium chloride provided with the kit and never faced this problem.
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques