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

70 Bartlett
2. 5× TBE: 54 g of Tris-base, 27.5 g of boric acid, 20 mL of 0.5 M EDTA (pH 8.0) dissolved
in 800 mL distilled water; make up 1 L.
3. 10% w/v ammonium persulphate (make fresh for each use).
4. TEMED (Bio–Rad).
5. Gel loading buffer: 0.25% Bromophenol blue (w/v), 0.25% xylene cyanol FF (w/v), and
30% glycerol (v/v) in distilled water (see Note 1).
6. Molecular weight markers (e.g., 100-bp ladder; Gibco).
7. Vertical gel electrophoresis system, spacers (1.0 mm; e.g., Bio–Rad Protean II).
3.1.2.2. DENATURING PAGE
1. 40% Acrylamide (38 g of acrylamide, 2 g of bis-acrylamide, Bio–Rad, UK) in distilled
water.
2. 5× TBE: 54 g of Tris-base, 27.5 g of boric acid, 20 mL of 0.5 M EDTA (pH 8.0) dissolved
in 800 mL of distilled water; make up to 1 L.
3. Urea (Sigma, UK).
4. 10% w/v ammonium persulphate (make fresh for each use).
5. Temed (Bio–Rad).
6. Radioactively labeled molecular weight markers: We have used Hinf1-digested plasmids
labeled with Klenow enzyme (see Note 3).
7. Gel loading buffer: 0.25% Bromophenol blue (w/v), 0.25% xylene cyanol FF (w/v), and
30% glycerol (v/v) in distilled water (see Note 1).
8. Vertical gel electrophoresis system, spacers (0.6 mm).
9. Gel fixative: Prepare 2 L of 10% acetic acid and 10% methanol in distilled water.
10. Whatmann 3MM Paper.
11. Gel dryer.
12. Autoradiographic film and cassettes.
13. Developer and fixative for above.
3.1.3. Recovery of DNA from Acrylamide Gels
1. Maxam & Gilberts solution: 0.5 M ammonium acetate, 0.1% sodium dodecyl sulphate,
and 1 mM EDTA; store at –20°C.
2. Transfer RNA (tRNA) 10 mg/mL and store at –20°C (optional).
3. Absolute ethanol (–20°C).
4. 70% ethanol (–20°C).
4. Methods
4.1. Agarose Gel Electrophoresis
1. Prepare a clean flat bed electrophoresis tray, with sealed ends (see Note 4) and a comb
with the appropriate number of samples. Fill tank with 1× TAE.
2. Dissolve 3 g of low melting point agar in 100 mL of 1× TAE buffer.
3. Weigh the flask and note weight before microwaving.
4. Microwave until clear, take care not to over boil (see Note 5).
5. Allow to cool slightly, weigh, and add sufficient distilled water to make up to premicrowave
weight.
6. Cool agarose to approx 50°C before pouring into gel support. Insert comb 0.5 to 1.0 mm
above the base of the tank, add agarose and allow to set (30 to 60 min).
7. Remove buffer dams or tape and place gel in electrophoresis tank. Add sufficient buffer
to cover gel to approx 1 mm.