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 #1Reaction #2Reaction #3Reaction #4Reaction #5Reaction #6
RNA2μL2μL2μL2μL2μL2μL
5x Nuclease Buffer5μL5μL5μL − protein buffer5μL − protein buffer5μL − protein buffer5μL − protein buffer
100mM MgCl2.5μL2.5μL2.5μL2.5μL2.5μL2.5μL
1M KCl1.25μL1.25μL1.25μL1.25μL1.25μL1.25μL
Protein+ Conc #1+ Conc #2+ Conc #3+ Conc #4
H2OTo 25μLTo 25μLTo 25μLTo 25μLTo 25μLTo 25μL
Time0 min60 min60 min60 min60 min60 min

Table #2

Reaction #1Reaction #2Reaction #3Reaction #4Reaction #5Reaction #6
RNA2μL2μL2μL2μL2μL2μL
5x Nuclease Buffer5μL5μL5μL − protein buffer5μL − protein buffer5μL − protein buffer5μL − protein buffer
100mM MgCl2.5μL2.5μL2.5μL2.5μL2.5μL2.5μL
1M KCl1.25μL1.25μL1.25μL1.25μL1.25μL1.25μL
Protein++++
H2OTo 25μLTo 25μLTo 25μLTo 25μLTo 25μLTo 25μL
Time0 min120 min15 min30 min60 min120 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
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