33 Identification of Cell Lines
333
4. After incubation, centrifug the tube at room temperature at 14,000 g
for 15 min.
5. Transfer the supernatant to another micro centrifuge tube, and add
270 f!l of 10 M ammonium acetate and 600 f!l isopropanol to the supernatant.
6. Turn the tube over several times, and keep the tube at -80°C for 20
min.
7. Thaw the sample to room temperature, and centrifuge the tube at
14,000 g for 15 min at room temperature.
8. Remove the supernatant and rinse the sediment with 1,000 f!l of 70%
ethanol.
9. Centrifuge the tube at 14,000 g for 15 min at room temperature.
10. Discard the supernatant.
11. Air-dry the resulting sediment under reduced pressure.
12. Dissolve the dried material in 50 f!l TE buffer, and store it as template
DNA at 4°C until use (see Comments).
1. In a micro centrifuge tube, mix 10 f!l of lOx Takara buffer, 8 f!l dNTP peR
mixture, 4 f!l primer (50 f!M), Takara Taq DNA polymerase (2.5 units)
and 1 f!l template DNA (the extracted DNA); add distilled water to
make up the total volume to 100 f!l.
2. Stir the mixture lightly.
3. Place the reaction mixture in a thermal cycler.
4. Run the PCR at 94°C for 2 min (denaturation step), then run 31 cycles,
each consisting of 94°C for 1 min, 42°C for 1 min, (annealing step) and
72°C for 2 min (extension step) and after that 72°C for 7 min, and
bring the temperature to 4°C. Keep the PCR product in a refrigerator
at 4°C.
1. Mix 8 f!l PCR product and 2 f!lloading buffer (3% glycerol, 30 mM Electrophoresis
EDTA, 0.03% bromophenol blue, and 0.03% xylene cyanol) gently. Mixing of small drops of PCR product and loading buffer is easily performed on a sheet If Parafilm.
2. Place the mixed drop on 1.4% agarose gel plate, and run electrophoresis
at 100V for 35 min.
3. After electrophoresis, stain the agar plate with 0.5 f!g/ml ethidium bromide in TAE buffer.
4. Record the DNA bands under UV illumination.
Comments
DNA strand is fragile. To avoid mechanical disintegration of DNA, it is
recommended to use a plastic chip whose tip is cut to enlarge the opening approximately 1 mm in diameter when DNA solution is handled.
Various DNA extraction kits are commercially available (e.g., Genera-
333
4. After incubation, centrifug the tube at room temperature at 14,000 g
for 15 min.
5. Transfer the supernatant to another micro centrifuge tube, and add
270 f!l of 10 M ammonium acetate and 600 f!l isopropanol to the supernatant.
6. Turn the tube over several times, and keep the tube at -80°C for 20
min.
7. Thaw the sample to room temperature, and centrifuge the tube at
14,000 g for 15 min at room temperature.
8. Remove the supernatant and rinse the sediment with 1,000 f!l of 70%
ethanol.
9. Centrifuge the tube at 14,000 g for 15 min at room temperature.
10. Discard the supernatant.
11. Air-dry the resulting sediment under reduced pressure.
12. Dissolve the dried material in 50 f!l TE buffer, and store it as template
DNA at 4°C until use (see Comments).
1. In a micro centrifuge tube, mix 10 f!l of lOx Takara buffer, 8 f!l dNTP peR
mixture, 4 f!l primer (50 f!M), Takara Taq DNA polymerase (2.5 units)
and 1 f!l template DNA (the extracted DNA); add distilled water to
make up the total volume to 100 f!l.
2. Stir the mixture lightly.
3. Place the reaction mixture in a thermal cycler.
4. Run the PCR at 94°C for 2 min (denaturation step), then run 31 cycles,
each consisting of 94°C for 1 min, 42°C for 1 min, (annealing step) and
72°C for 2 min (extension step) and after that 72°C for 7 min, and
bring the temperature to 4°C. Keep the PCR product in a refrigerator
at 4°C.
1. Mix 8 f!l PCR product and 2 f!lloading buffer (3% glycerol, 30 mM Electrophoresis
EDTA, 0.03% bromophenol blue, and 0.03% xylene cyanol) gently. Mixing of small drops of PCR product and loading buffer is easily performed on a sheet If Parafilm.
2. Place the mixed drop on 1.4% agarose gel plate, and run electrophoresis
at 100V for 35 min.
3. After electrophoresis, stain the agar plate with 0.5 f!g/ml ethidium bromide in TAE buffer.
4. Record the DNA bands under UV illumination.
Comments
DNA strand is fragile. To avoid mechanical disintegration of DNA, it is
recommended to use a plastic chip whose tip is cut to enlarge the opening approximately 1 mm in diameter when DNA solution is handled.
Various DNA extraction kits are commercially available (e.g., Genera-