Bulk tRNAs were prepared as previously described using acid buffered-phenol (phenol saturated with 50 mM sodium acetate, pH 5.8) and alcohol precipitation [20 (link)]. Nucleosides were prepared as described in [61 (link)] by hydrolyzing bulk tRNA with 10 units of Nuclease P1 (Sigma) overnight at 37°C, with the addition of 0.01 units of phosphodiesterase I (Sigma) and 3 µL E. coli alkaline phosphatase (Sigma). The hydrolyzed nucleosides were further purified by filtering through a 5 kD MWCO filter (Millipore) (to remove enzymes), dried in a CentriVap Concentrator, and suspended in 20 µL of water prior to analysis by HPLC or LC-MS/MS.
Protocol detail
Bulk tRNA Isolation and Nucleoside Analysis
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Acid
Alcohol
Alkaline phosphatase
Bulk
E coli
Enzymes
Hplc
Ms ms
Nucleosides
Phenol
Phosphodiesterase i
Sodium acetate
Trna
Corresponding Organization :
Other organizations : University of Florida, Institut de Biologie Intégrative de la Cellule, Institut thématique Génétique, génomique et bioinformatique, MRC Laboratory of Molecular Biology
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Variable analysis
independent variables
- Preparation of bulk tRNAs using acid buffered-phenol and alcohol precipitation
- Hydrolysis of bulk tRNA with Nuclease P1, phosphodiesterase I, and E. coli alkaline phosphatase
- Nucleosides obtained from the hydrolysis of bulk tRNA
- Overnight incubation at 37°C
- Filtering through a 5 kD MWCO filter to remove enzymes
- Drying in a CentriVap Concentrator
- Suspending the hydrolyzed nucleosides in 20 µL of water prior to analysis by HPLC or LC-MS/MS
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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