RNase Free Solutions for RNA-FISH
(by mari_ferrarini from hotmail.com)
Thu Feb 4 11:12:45 EST 2010
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.
Thank you a lot for the patience and time!!
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