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

Direct Automated Cycle Sequencing 343
1. Aliquot an appropriate amount of the PCR into a 0.6-mL microfuge tube, and dilute to
a total of 20 µL with distilled water.
2. Add 20 µL of 4 M ammonium acetate into the microfuge tube, mixing well.
3. Add 40 µL of isopropanol into the tube, mix well, leave at room temperature for 10 min,
and centrifuge the microfuge tube for 10 min at 12,000g.
4. Carefully remove the supernatant and wash the pellet with 70% (v/v) ethanol. Then, briefly
dry the pellet under vacuum.
5. Resuspend the pellet in 20 µL of TE buffer.
3.2. Cycle Sequencing of PCR Products
The amount of PCR product should be estimated on an agarose gel before sequencing.
Approximately 1 µg of double-stranded DNA template or 0.5 µg of single-stranded
DNA template is required for each sequencing reaction.
1. Mix the following reagents in a 0.6-mL microfuge tube: 5 mL of DNA template, 1 µL
of primer (from a 3.2-pmol stock solution), and 4.5 µL of sterile dH 2 O (see Notes 4
and 5).
2. Add 9.5 µL of ABI Prism ready reaction DyeDeoxy terminator premix.
3. Spin briefly to collect the reaction mix in the bottom of the tube and overlay with approx
50 µL of mineral oil.
4. Place the tubes in a thermal cycler (Perkin–Elmer Cetus [Warrington, UK] model 480 or
9600) that has been preheated to 96°C.
5. Immediately begin the cycle sequencing program, which is as follows: rapid thermal ramp
to 96°C; 96°C for 30 s; Rapid thermal ramp to 50°C; 50°C for 15 s; Rapid thermal ramp
to 60°C; and 60°C for 4 min for a total of 25 cycles.
6. Try to keep the samples in the dark at 4°C until further processing because they are
sensitive to light.
7. Remove the excess DyeDeoxy terminators from the completed sequencing reactions.
3.3. PhenolChloroform Extraction of Cycle Sequencing Products
This step is essential to remove excess primers and unincorporated nucleotides.
1. To each sample, add 80 µL of sterile dH 2 O.
2. Either add 100 µL of chloroform to dissolve the oil or remove the oil with a pipet.
3. Add 100 µL of phenolH 2 Ochloroform (681814) to the sample and mix well by
vortexing.
4. Centrifuge the sample at 12,000g for 1 min. Remove and discard the lower organic phase.
5. Re-extract the aqueous layer and transfer the aqueous upper layer to a clean tube.
6. Add 15 µL of 2 M sodium acetate and 300 µL of 100% ethanol to precipitate the extension
products (see Note 7).
7. Centrifuge at 12,000g for 15 min at room temperature.
8. Carefully remove the supernatant and wash the pellet with 70% ethanol. Then, briefly dry
the pellet under a vacuum (see Note 2).
3.4. Preparation of Samples for Loading
1. Add 4 µL of deionized formamide: 50 mM EDTA, pH 8.0 (51), to each sample tube, and
mix well to dissolve the dry pellet.
2. Centrifuge briefly to collect the liquid at the bottom of the tube.
3. Before loading, heat the samples at 90°C for 2 to 3 min to denature. Then, transfer
immediately onto ice.