On Feb 4, 11:12 am, Mari Ferrarini <[email protected]> wrote: > Hello all, > > I've recently started doing the RNA-FISH technique but I have some doubts > about the reagents... > > Here's a simple list of all reagents I need to use and all of them need to be > RNase free. I'm sorry I may sound a little newbie (that's because I am! LOL): > > 1. PBS 10X (Treated with DEPC and autoclaved afterwards); > 2. SSC 20X (Treated with DEPC and autoclaved afterwards - I know that in this > concentration the enzyme is not active, but I'm willing to use it as a final > concentration of 2X, 1X and 0.5X); > 3. HCl 2M and 0.2M (I don't have a stock of HCl RNase free, so I don't know > if it is necessary - or possible - to use DEPC and autoclave it afterwards); > 4. PFA 4% in PBS1X (Also don't know if treatment is possible); > 5. Formamide 50% in SSC2X (No clue!); > 6. 0,5% Saponin; 0,02% BSA (I can't autoclave it, so can I add the BSA after > treating the solution?) > 7. Proteinase K 1ug/ml in 500mM NaCl and 10mM Tris (This one, I'm sure I > can't treat with DEPC, any alternatives?) > > Is it possible to treat them with DEPC and autoclave them? Am I doing > anything wrong? The water I use is RNase free and also the tips, tubes and > glassware. > >
I stopped using DEPC for RNA isolation (well, for anything, really) years ago. Use good water, clean reagents, and keep your fingers out your samples and you should be fine. For sure, you don't need to treat PFA, formamide, HCl, or Proteinase K with anything to make them RNase-free. Nick -- Nick Theodorakis [email protected] contact form: http://theodorakis.net/contact.html _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
