183
4. Buffer A: 20 mM Tris–HCl, 50 mM NaCl at pH 8.0.
5. Buffer B: 20 mM Tris–HCl, 1 M NaCl at pH 8.0.
1. Complexation buffer: 50 mM sodium phosphate, 100 mM
NaCl at pH 7.0.
2. 2× sample buffer: 125 mM Tris–HCl (pH 6.8), 4% (w/v) SDS,
20% (v/v) glycerol, 0.01% bromophenol blue.
3. 30% (w/v) acrylamide/bisacrylamide solution (29:1, w/w).
4. N,N,N′,N′-tetramethylethylenediamine (TEMED).
5. 10% (w/v) ammonium persulfate solution in water.
6. Isopropanol.
7. Running buffer: 187 mM glycine, 19 mM Tris–HCl, 3.5 mM
SDS.
8. Protein molecular weight marker, prestained.
9. Coomassie Brilliant Blue.
1. PBS
+
: 50 mM sodium phosphate, 100 mM NaCl, 1 mg/mL
bovine serum albumin (BSA) at pH 7.0.
2. 50 μM ssDNA template stock solution dissolved in H 2 O
(Table 1).
3. 50 μM ssDNA target stock solution dissolved in H 2 O
(Table 1).
4. 500 μM nitrocefin, chromogenic β-lactamase substrate (e.g.,
EMD Millipore, VWR, Cat No: 80,017–707) dissolved in
PBS.
5. Multiwell plate reader (with absorbance mode).
6. Transparent 96-well plates.
1. PBS
+
: 50 mM sodium phosphate, 100 mM NaCl, 1 mg/mL
BSA at pH 7.0.
2. 50 μM ssDNA template stock solution dissolved in H 2 O
(Table 1).
3. 50 μM ssDNA target stock solution dissolved in H 2 O (Table 1).
4. 20 μM CCF2-FA, fluorescent β-lactamase substrate (e.g.,
Invitrogen, Cat No: K1034) dissolved in PBS.
5. Multiwell plate reader (with fluorescence mode).
6. Black 96-well plate.
2.4 Semi-Native
SDS-PAGE
of Reconstituted
DNA-Directed EnzymeInhibitor Complex
2.5 Enzyme Activity
Assay of Reconstituted
DNA-Directed EnzymeInhibitor Complex
2.6 Enzyme Activity
Assay at Different
Input Oligonucleotide
Concentrations
DNA Sensors Based on β-Lactamase