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RNase Protection Assay for RNA Modification Detection References: Current Protocols, Chapter 4.7.1
 

Summary: 62
RNase Protection Assay for RNA Modification Detection
References: Current Protocols, Chapter 4.7.1
Sambrook, Fritsch and Maniatis, Chapter 7.71
Segal and Eichler, 1991, JBC 266(36):24385-9.
Buffers:
Denaturation Buffer: 2 Parts freshly thawed formamide (40% final)
1 Part 5X Denaturation Buffer
2 Parts sterile ddH2O
5X Denaturation Buffer: 100 mls
200 mM PIPES, pH 6.4 6.93 g (pH with HCl)
2 M NaCl 11.69 g
5 mM EDTA 0.19 g (Do not add from stock) Treat with DEPC
Digestion Buffer: 1 ml from Stocks
300 mM NaCl 60 l of 5M NaCl
10 mM Tris-HCl, pH 7.5 10 l of 1 M Tris-Cl, pH 7.5
5 mM EDTA 10 l of 0.5 M EDTA
920 l ddH2O
Freshly add: RNase A to 20 g/ml (1/500 of 10 mg/ml stock; Sigma R-5000)
RNase T1 to 3 g/ml (1/500 of 1.5 mg/ml stock; Sigma R-1003)

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine