29 Spectral Imaging Analysis of Metaphase and Interphase Cells
373
2. Pellet the yeast cells from 5 ml AHC medium at 400 rpm for 6 min.
Resuspend cells in 0.5 rnl EDTA (0.125 M), pH 7.8. Spin down again
and remove the supernatant.
3. Add 500 ~l of SCE to a 70 ~l pellet and resuspend. Mix with an equal
volume of 1.5% LMP agarose at 43°C, quickly pipette up and down and
vortex for 1-2 s to resuspend. Pipette into molds (BioRAD) and allow to
solidify.
4. Remove plugs from molds and incubate samples in 2 ml SCE with 100
~l of zymolase (10 mg/ml) and shake at ISO rpm at 30°C for 2.5 h to
overnight.
5. Remove SCE and add 2 ml ofES with 100 ~l of proteinase K (20 mg/ml).
Shake 5 h to overnight at 50°e.
6. Remove ES and rinse 5 times with 6 ml of TE50 for 30 min each rinse.
Plugs are now ready for running PFGE or to be stored at 4°e.
7. PFGE running conditions: voltage gradient, 6 V/cm; switching time
ramp, 79-94 s; running time, 38 h; agarose concentration, 1.0%
LMP agarose; running temperature, l4°C; running buffer, O.5xTBE.
8. Stain the gel with ethidium bromide (O.5llg/ml in H 2 0) and cut out the
target band. Transfer gel slice to a clean tube.
9. Wash gel slices with water for 30 min, and then wash with I x p-agarose
buffer for 30 min.
10. Melt the gel completely by heating for 10 min at 85°e. Transfer the
molten agarose to 43°C water bath. Add 1 ~l p-agarase for every 25
~I molten agarose and continue incubation for 2 h. The sample can
now be used for the DOP-PCR reactions or stored at 4°e.
There are two different DNA polymerase chain reactions, T7 DNA polymerase and Taq polymerase. The initial seven cycles of PCR use primer
JUNI annealed at 37°C and extended by T7 DNA polymerase. The PCR
products are then further amplified at higher annealing temperature
(50°e) with primer JUNI5 and Taq polymerase for 35 cycles.
T7 DNA polymerase (7 cycles) PCR:
1. Prepare the T7 Mix (total 27 Ill): 19.0 III water, 5.2 ~l 5x Sequenase
reaction buffer, 0.81114 dNTP, 0.6111 primer JUNI, and 0.4111 Sequenase
II (T7 DNA polymerase).
Pulsed field gel
electrophoresis
(PFGE
Degenerate oligonucleotide-primed
PCR (DOP-PCR)
373
2. Pellet the yeast cells from 5 ml AHC medium at 400 rpm for 6 min.
Resuspend cells in 0.5 rnl EDTA (0.125 M), pH 7.8. Spin down again
and remove the supernatant.
3. Add 500 ~l of SCE to a 70 ~l pellet and resuspend. Mix with an equal
volume of 1.5% LMP agarose at 43°C, quickly pipette up and down and
vortex for 1-2 s to resuspend. Pipette into molds (BioRAD) and allow to
solidify.
4. Remove plugs from molds and incubate samples in 2 ml SCE with 100
~l of zymolase (10 mg/ml) and shake at ISO rpm at 30°C for 2.5 h to
overnight.
5. Remove SCE and add 2 ml ofES with 100 ~l of proteinase K (20 mg/ml).
Shake 5 h to overnight at 50°e.
6. Remove ES and rinse 5 times with 6 ml of TE50 for 30 min each rinse.
Plugs are now ready for running PFGE or to be stored at 4°e.
7. PFGE running conditions: voltage gradient, 6 V/cm; switching time
ramp, 79-94 s; running time, 38 h; agarose concentration, 1.0%
LMP agarose; running temperature, l4°C; running buffer, O.5xTBE.
8. Stain the gel with ethidium bromide (O.5llg/ml in H 2 0) and cut out the
target band. Transfer gel slice to a clean tube.
9. Wash gel slices with water for 30 min, and then wash with I x p-agarose
buffer for 30 min.
10. Melt the gel completely by heating for 10 min at 85°e. Transfer the
molten agarose to 43°C water bath. Add 1 ~l p-agarase for every 25
~I molten agarose and continue incubation for 2 h. The sample can
now be used for the DOP-PCR reactions or stored at 4°e.
There are two different DNA polymerase chain reactions, T7 DNA polymerase and Taq polymerase. The initial seven cycles of PCR use primer
JUNI annealed at 37°C and extended by T7 DNA polymerase. The PCR
products are then further amplified at higher annealing temperature
(50°e) with primer JUNI5 and Taq polymerase for 35 cycles.
T7 DNA polymerase (7 cycles) PCR:
1. Prepare the T7 Mix (total 27 Ill): 19.0 III water, 5.2 ~l 5x Sequenase
reaction buffer, 0.81114 dNTP, 0.6111 primer JUNI, and 0.4111 Sequenase
II (T7 DNA polymerase).
Pulsed field gel
electrophoresis
(PFGE
Degenerate oligonucleotide-primed
PCR (DOP-PCR)