John M. S. Bartlett.pdf - Bio-Nica.info

264 Case-Green, Pritchard, and Southern
2.3. Hybridization
2.3.1. Hybridization Assay
1. Buffer: 1 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.01% SDS.
2. Petri dish.
3. Tissues.
4. Forceps.
5. Strip cut from oligonucleotide array (30 × 2 mm).
6. Target single-stranded RNA (see Subheading 3.2.1.).
2.3.2. Hybridization of Target for Polymerase Assay
1. Buffer: 1 M NaCl.
2. Petri dish.
3. Tissues.
4. Forceps.
5. Strip cut from oligonucleotide array (30 × 2 mm).
6. Target single-stranded DNA (see Subheading 3.2.2.).
2.3.3. Hybridization of Target and Reporter for Ligation Assays
1. Buffer: 1 M NaCl.
2. Petri dish.
3. Tissues.
4. Forceps.
5. Labeled reporter oligonucleotide containing 5′ phosphate group.
6. Target single-stranded DNA (see Subheading 3.2.2.).
7. Strip of oligonucleotide array (30 × 2 mm).
2.4. Enzyme-Aided Assays
2.4.1. Ligation
1. 5µ ligation buffer: 100 mM Tris-HCl, pH 8.3, 0.5% Triton X-100, 50 mM MgCl 2 , 250 mM
KCl, 5 mM NAD + , 50 mM DTT, 5 mM EDTA (Advanced Biotechnologies Ltd.).
2. Thermus thermophilus DNA ligase (Tth DNA ligase; Advanced Biotechnologies Ltd).
3. Petri dish set up as humid chamber.
4. Forceps.
2.4.2. Primer Extension with Polymerase
1. 5µ Polymerase buffer: 200 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 250 mM NaCl
(Amersham).
2. Polymerase dilution buffer: 50 mM Tris-HCl, pH 7.5; 10 mM DTT (Amersham).
3. DTT (100 mM, Promega).
4. T7 Sequenase V2. 13 U/µL (Amersham).
5. Dideoxynucleotidetriphosphate [α- 33 P]ddNTP (450 µCi/mL; Amersham).
6. Humid chamber.
7. Forceps.
8. Buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.