I am using this paper for RNA extraction from Arabidopsis seeds:
Suzuki Y, Kawazu T, Koyama H. RNA isolation from siliques, dry seeds, and other tissues of Arabidopsis thaliana. BioTechniques. 2004 Oct;37(4):542, 544
The paper asks to prepare phenol mix of ''water-saturated phenol containing 35% (w/v) of guanidium thiocyanate''. I tried to dissolve 7g guanidium thiocyanate in (~20 ml) lower layer of water-saturated phenol for an hour. It did not dissolve. I found that guanidium thiocyanate stocks are usually made in Tris with heating to 65C for solubilization. Heating did not help me either.
Can anyone guide me to prepare this mix? Thanks!
You can't dissolve GuSCN in phenol, even water-saturated. Maybe the protocol in BioTechniques lacks details. You can contact directly the corresponding author, hoping he's still in activity and his e-mail is still valid ([email protected]). In the original protocol, GuSCN and phenol are used sequentially, not in mixture. You can have a look at the Nature Protocols paper, in which how to make reagents and buffer is well described.
Another way to dissolve GuSCN in phenol is to first
- Remove the aqueous layer on the surface of the water saturated phenol whilst dissolving GuSCN together with ammonium thiocynate, sodium acetate and glycerol in autoclaved water.
Then you can add your phenol to the resultant mixture.
I have tried that several times and it works. All the best
Thank You guys for your help. The protocol also asked to add sodium acetate. After I added that, guanidinium thiocyanate dissolved!
- Badges
- Science topic
- Similar topics
- Methodology
- Research Methodology
- Research Papers