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

Digoxigenin 349
pH 8.0, 50 mM MgCl 2 ); loading buffer containing formamide; and Termination mixtures
containing dNTPs and the appropriate ddNTP:
ddATP (dATP, dCTP, dGTP, and dTTP, 25 µM each; 850 µM ddATP; 950 µM MgCl 2 ;
pH 7.5).
ddCTP (dATP, dCTP, dGTP, dTTP, 25 µM each; 400 µM ddCTP; 500 µM MgCl 2 ;
pH 7.5).
ddGTP (dATP, dCTP, dGTP, dTTP, 25 µM each; 75 µM ddGTP; 175 µM MgCl 2 ;
pH 7.5).
ddTTP (dATP, dCTP, dGTP, dTTP, 25 µM each; 1275 µM ddTTP; 1370 µM MgCl 2 ;
pH 7.5).
A second set of termination mixtures is shipped with the kit substituting 7-deaza-dGTP
for dGTP.
4. 0.5-mL thin-walled reaction tubes (N801-0537, Applied Biosystems, Foster City, CA).
5. Eppendorf centrifuge.
6. Thermal cycler for cycle sequencing (see Note 6 for reference).
2.3. Preparation of Sequencing Gel
1. 10× TBE: 0.9 M Tris, pH 8.3, 0.9 M boric acid, and 25 mM EDTA (6).
2. 10% Ammonium persulfate (w/v) (should be prepared freshly before use).
3. Sigmacote (SL-2, Sigma).
4. GelMix 8 (#5545UA, Gibco BRL, Gaithersburg, MD), containing 7.6% acrylamide (w/v),
0.4% N,N′-methylene bis-acrylamide, 7.0 M urea, 100 mM Tris-borate, pH 8.3, 1 mM
Na 2 EDTA, 3 mM TEMED.
5. 10-mL disposable syringes with needles.
6. Scotch electrical tape 50 mm (FE-5000-0409-1, 3 M).
7. Plastic foil (e.g., Saran Wrap).
2.4. Gel Electrophoresis and Visualization of Sequencing Results
1. 1× Tris-buffered saline (TBS) 50 mM Tris-HCl, pH 7.5, 150 mM NaCl.
2. Alkaline phosphatase reaction buffer: 0.1 M Tris-HCl, pH 9.5, 50 mM MgCl 2 ,
0.1 M NaCl.
3. Stock solutions of 70% (v/v) and 100% N,N-dimethyl formamide.
4. Digoxigenin Detection Kit for Glycoconjugate and Protein Analysis (#1210220, Boehringer
Mannheim), containing: antidigoxigenin antibodies conjugated to alkaline phosphatase;
blocking reagent (purified casein fraction); nitroblue tetrazoliumchloride, 77 mg/mL in
70% N,N-dimethyl formamide; and X-Phosphate (5-bromo-4-chloro-3-indolylphosphate,
4-toluidinium salt), 50 mg/mL in 100% N,N-dimethyl formamide.
5. Nylon membrane, positively charged (#1417240, Boehringer Mannheim).
6. Whatman chromatography paper (#3030917, 3MM Whatman International Ltd., Maidstone,
UK).
7. Plastic hybridization bags.
8. Scalpel blade.
9. Standard sequencing equipment: sequencing apparatus (Model S2, Gibco BRL) and
high-voltage power supply (PS 9009, Gibco BRL).
10. Ultraviolet transilluminator (302 nm) for DNA crosslinking (Hoefer).
11. Eppendorf centrifuge.
12. Heating block or thermal cycler for denaturation of DNA.
13. Electric sealer for closing hybridization bags.