Read Lab: Researching Trypanosoma brucei | Protocols

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.

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