332
Related Techniques
- Thermal cycler
- Deep freezer
- Incubator or heat block
- Agarose gel electrophoresis apparatus (e.g., Mupid-2, Cosmo Bio)
- UV illuminator with a recording system
- Camera (a Polaroid camera is preferable)
- Pipettes
- Pipette aid
- Pipetman (1000,200,20, and 2f1l; Gilson)
- Pasteur pipettes
- Pipette chip (0.1-2, 1-200, and 101-1000fil; Molecular Bio)
- PCR tube (0.2 ml)
Solutions - Carlson's solution (see Appendix 1)
- Lytic buffer (200 mM Tris-HCI pH 8.0,70 mM Na 2 EDTA,2 M NaCI,20
mM sodium metabisulfite), TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM
Na 2 EDTA), TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM Na 2
EDTA), Takara buffer (Takara), dNTP mixture (2.5 mM each; Takara),
Takara taq DNA polymerase (Takara), primer (5'-CCG CAT CTA C-3',
synthesized by Nisseiken)
Chemicals - Sarcosyl
- Isopropanol
lr1iscellaneous - Parafilm (American National Can)
Preparation
of cells
Cell
washing
DNA
extraction
Procedure
1. Culture cells, taking care to avoid mycoplasmal contamination.
2. Harvest cells by suspending the cells in the culture medium and centrifuging the suspension when the growth of cells has reached a plateau (between 2 x 10 6 and 3 x 10 7 cells/ml cells are required).
1. Suspend the cell pellet in 1 ml Carlson's solution, and then centrifuge
the suspension at 150 g for 5 min.
2. Suspend the cell pellet in 1.5 ml Carlson's solution, and transfer it to a
1.5 ml micro centrifuge tube.
3. Centrifuge the suspension at 220 g for 5 min.
4. Discard the supernatant.
1. Add 480 fil of lytic buffer to the sediment in the micro centrifuge tube
keeping the tube in flaked ice, and then turn the tube over five times.
2. Add a further 120 fil of 5% sodium sarcosyl aqueous solution to the
tube, and turn the tube over several times.
3. Incubate the tube in an incubator or a heat block at 55°C for 2 h.
Related Techniques
- Thermal cycler
- Deep freezer
- Incubator or heat block
- Agarose gel electrophoresis apparatus (e.g., Mupid-2, Cosmo Bio)
- UV illuminator with a recording system
- Camera (a Polaroid camera is preferable)
- Pipettes
- Pipette aid
- Pipetman (1000,200,20, and 2f1l; Gilson)
- Pasteur pipettes
- Pipette chip (0.1-2, 1-200, and 101-1000fil; Molecular Bio)
- PCR tube (0.2 ml)
Solutions - Carlson's solution (see Appendix 1)
- Lytic buffer (200 mM Tris-HCI pH 8.0,70 mM Na 2 EDTA,2 M NaCI,20
mM sodium metabisulfite), TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM
Na 2 EDTA), TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM Na 2
EDTA), Takara buffer (Takara), dNTP mixture (2.5 mM each; Takara),
Takara taq DNA polymerase (Takara), primer (5'-CCG CAT CTA C-3',
synthesized by Nisseiken)
Chemicals - Sarcosyl
- Isopropanol
lr1iscellaneous - Parafilm (American National Can)
Preparation
of cells
Cell
washing
DNA
extraction
Procedure
1. Culture cells, taking care to avoid mycoplasmal contamination.
2. Harvest cells by suspending the cells in the culture medium and centrifuging the suspension when the growth of cells has reached a plateau (between 2 x 10 6 and 3 x 10 7 cells/ml cells are required).
1. Suspend the cell pellet in 1 ml Carlson's solution, and then centrifuge
the suspension at 150 g for 5 min.
2. Suspend the cell pellet in 1.5 ml Carlson's solution, and transfer it to a
1.5 ml micro centrifuge tube.
3. Centrifuge the suspension at 220 g for 5 min.
4. Discard the supernatant.
1. Add 480 fil of lytic buffer to the sediment in the micro centrifuge tube
keeping the tube in flaked ice, and then turn the tube over five times.
2. Add a further 120 fil of 5% sodium sarcosyl aqueous solution to the
tube, and turn the tube over several times.
3. Incubate the tube in an incubator or a heat block at 55°C for 2 h.