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

Serial Analysis of Gene Expression 275
2.7. Gel Materials
1. 40% polyacrylamide (37.51 acrylamidebis) (Bio–Rad Ltd, Hertfordshire, UK).
2. 40% polyacrylamide (191 acrylamidebis) (Bio–Rad).
3. DNA molecular weight markers, for example, 10- and 100-bp DNA ladders (Invitrogen).
4. SYBR ® Green I nucleic acid gel stain (Molecular Probes Inc., OR) or a 0.5 µg/mL solution
of Ethidium Bromide (Sigma).
5. Gel-loading buffer containing Bromophenol Blue and Xylene Cyanol (e.g., Ambion).
6. Apparatus for vertical polyacrylamide gel electrophoresis, for example, Atto Maxi Slab
with 16- × 16-cm glass gel plates, 1.5-mm spacers, and 12- and 20-well combs (Genetic
Research Instrumentation, Essex, UK).
7. 50× Tris-Acetate buffer: 242 g of Tris base, 57.1 mL of glacial acetic acid, and 100 mL of
0.5 M EDTA (pH 8.0) made up to 1 L with dH 2 O.
8. 10% Ammonium persulfate.
9. TEMED (N,N,N′,N′-Tetramethylethylenediamine; Sigma).
10. Sterile scalpel.
11. 21-gauge needles.
12. Spin-X filter microcentrifuge tubes (Corning Ltd, Buckinghamshire, UK).
13. Materials, buffers, and apparatus for agarose gel electrophoresis
2.8. Polymerase Chain Reaction (PCR)
1. Option 1: Johns Hopkins protocol. PLATINUM Taq DNA Polymerase (5 U/µL; Invitrogen);
10× PCR buffer: 166 mM (NH 4 ) 2 SO 4 , 670 mM Tris-HCl (pH 8.8), 67 mM MgCl 2 , 100 mM
β-mercaptoethanol; Dimethyl sulphoxide (Sigma). Option 2: own modification. HotStarTaq
DNA Polymerase Kit and Taq PCR core kit (Qiagen).
2. dNTPs (10 mM; Invitrogen; included in some kits).
3. Thermal cycler of choice (e.g., Thermo Hybaid, Middlesex, UK).
4. Mineral oil (Sigma). Ensure that it is free from PCR contamination.
2.9. Cloning
1. For cloning of concatemers: Zero Background Cloning Kit (Invitrogen).
2. ELECTROMAX DH10B Cells (Invitrogen).
3. S.O.C. Medium (Invitrogen).
4. Low-salt LB medium and agar plates with Zeocin , prepared according to instructions
in Zero Background Cloning Kit.
5. Electroporator and electroporation cuvettes (Thermo Hybaid).
2.10. Sequencing
1. Sequencing as performed locally, for example, use BigDye Primer Kit and ABI automated
sequencer (both Perkin–Elmer from Applied Biosystems, Warrington, UK).
2.11. SAGE Bioinformatics
Johns Hopkins SAGE program (downloaded from web site after registration), in
combination with NCBI’s GenBank ® databases and Microsoft ® Access and Excel
programs, or other software (see Subheading 1., last paragraph, and Table 1).
3. Methods (see Notes 1 and 2)
3.1. mRNA Preparation
1. Prepare total RNA from cells or tissue by standard methods, for example, TRIZOL
Reagent.