Degradation Assay
5x Nuclease Buffer | 100mM MgCl | 1M KCl |
---|---|---|
100mM Tris (pH=7.5) | ||
250mM KCl | ||
5mM EDTA | ||
50% Glycerol | ||
5mM DTT (Add fresh before experiment) |
For testing serial dilution protein concentrations in a degradation assay assemble reactions as in Table #1 below. If using one concentration of protein for a time course use Table #2 below. First 2 reactions are negative controls without protein. For EMP degradation assays only: add 2.5ul 10mM UTP per 25ul reaction.
Final concentrations of solutions in reactions are 1x Nuclease Buffer (20mM Tris (pH=7.5), 50mM KCl, 1mM EDTA, 10%glycerol, 1mM DTT), MgCl=10mM (note- concentration can be changed depending on enzyme), KCl=50mM, UTP ( if using EMP)=1mM.
Dilute RNA to required concentration so that 2ul can be added to each reaction; body labeled P32 RNA use 2.5 pmoles/reaction (1.25 pmoles/ul), 5' end labeled RNA oligos the amounts can vary depending on what you are looking for but 50,000cpm/reaction (25,000cmp/ul) was useful for testing recombinant RNaseD
If protein is resuspended in a buffer (ie. 1x Nuclease Buffer), that amount must be subtracted from the amount of Nuclease buffer to be added in a reaction to keep final concentrations the same in all reactions.
Table #1
Reaction #1 | Reaction #2 | Reaction #3 | Reaction #4 | Reaction #5 | Reaction #6 | |
RNA | 2μL | 2μL | 2μL | 2μL | 2μL | 2μL |
5x Nuclease Buffer | 5μL | 5μL | 5μL − protein buffer | 5μL − protein buffer | 5μL − protein buffer | 5μL − protein buffer |
100mM MgCl | 2.5μL | 2.5μL | 2.5μL | 2.5μL | 2.5μL | 2.5μL |
1M KCl | 1.25μL | 1.25μL | 1.25μL | 1.25μL | 1.25μL | 1.25μL |
Protein | − | − | + Conc #1 | + Conc #2 | + Conc #3 | + Conc #4 |
H2O | To 25μL | To 25μL | To 25μL | To 25μL | To 25μL | To 25μL |
Time | 0 min | 60 min | 60 min | 60 min | 60 min | 60 min |
Table #2
Reaction #1 | Reaction #2 | Reaction #3 | Reaction #4 | Reaction #5 | Reaction #6 | |
RNA | 2μL | 2μL | 2μL | 2μL | 2μL | 2μL |
5x Nuclease Buffer | 5μL | 5μL | 5μL − protein buffer | 5μL − protein buffer | 5μL − protein buffer | 5μL − protein buffer |
100mM MgCl | 2.5μL | 2.5μL | 2.5μL | 2.5μL | 2.5μL | 2.5μL |
1M KCl | 1.25μL | 1.25μL | 1.25μL | 1.25μL | 1.25μL | 1.25μL |
Protein | − | − | + | + | + | + |
H2O | To 25μL | To 25μL | To 25μL | To 25μL | To 25μL | To 25μL |
Time | 0 min | 120 min | 15 min | 30 min | 60 min | 120 min |
Incubate reactions at room temp for the indicated Time (length of Time can vary depending on the protein(s) used but 60 min works well for an initial test). Stop reactions with 5ul of 50mM EDTA/0.2% SDS. Add 30ul of phenol/chloroform low pH (for RNA only), tap with finger, and spin 5 min at 13,000rpm. Pipet 20ul of top layer into a new tube with 20ul 90% formamide dye, store in the -20°C or run on acrylamide/urea gel.
Download Protocol: Degradtion Assay
(note requires Microsoft Word to open)