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

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to sequence a 500-bp PCR fragment on an automated DNA sequencer with 99.3%
accuracy (10–12).
This chapter focuses on the techniques involved with the direct sequencing reactions
rather than on the use of the machine because each automated DNA sequencer will be
provided with an extensive manual for its operation.
2. Materials
All solutions should be made to the standard required for molecular biology. Use
molecular-biology-grade reagents and sterile distilled water. The reagents for the cycle
sequencing are available commercially.
2.1. Purification of PCR Products Before Cycle Sequencing
1. Ammonium acetate (4 M).
2. Isopropanol.
3. 70% (v/v) ethanol.
4. Tris-HCl (10 mM, pH 7.5), 1 mM EDTA.
2.2. Cycle Sequencing
Prism ready reaction DyeDeoxy terminator premix (1000 µL, Applied Biosystems
[ABI]) consists of 1.58 mM A-dyedeoxy, 94.74 µM T-dyedeoxy, 0.42 µM G-dyedeoxy,
47.37 µM C-dyedeoxy, 78.95 µM dITP, 15.79 µM dATP, 15.79 µM dCTP, 15.79 µM
dTTP, 168.42 mM Tris-HCl (pH 9.0), 4.21 mM (NH 4 ) 2 SO 4 , 42.1 mM MgCl 2 , 0.42 U/µL
AmpliTaq DNA polymerase.
2.3. Purification of PCR Products After Cycle Sequencing
1. Chloroform.
2. PhenolH2Ochloroform (161814) at room temperature.
3. Sodium acetate (2 M, pH 4.5).
4. 100 and 70% (v/v) ethanol at room temperature.
2.4. 6% Polyacrylamide Sequencing Gels
1. 10× TBE: 890 mM Tris-borate, 890 mM boric acid, and 20 mM EDTA, pH 8.3.
2. Urea (40 g).
3. 12 mL of 40% (w/v) acrylamide stock solution (191 acrylamide/bis-acrylamide).
4. dH 2 O (20 mL).
5. Mixed-bed ion-exchange resin (1 g).
6. TEMED.
7. 10% (w/v) ammonium persulfate, freshly made.
2.5. Loading Buffer
1. 50 mM EDTA, pH 8.0.
2. Deionized formamide.
3. Methods
3.1. Isopropanol Purification of PCR Products
It is essential to remove excess PCR primers before using DyeDeoxy terminators
for cycle sequencing (see Note 1).