The techniques described here were developed for the purpose of examining multiple time points during the passage of synchronized S. pombe cell populations through S phase. For this purpose it was necessary that clear, quantitative 2D gel patterns be obtained starting with relatively small quantities of cells. It was also important that the overall procedure be rapid and reproducible. Our previous experience with the BND-cellulose technique for enriching for replication intermediates suggested that the yield of replication intermediates could be variable. For this reason, and also because the BND-cellulose technique takes time (usually a full working day), we decided to skip the normal BND-cellulose enrichment step and rely on synchrony to provide sufficient enrichment of replication intermediates. Our experience to date suggests that this strategy offers a satisfactory degree of reproducibility and sensitivity (the results in the picture above are with single copy DNA). Although we haven't tried these procedures with S. cerevisiae cells, we suspect (based on our lab's experience with S. cerevisiae cells) that the procedures would work for them, too.
Preparation of Genomic DNA
200 ml yeast (S. pombe or S. cerevisiae) cells (0.81 X 10^7 cells/ml) are harvested by centrifugation at 5K rpm for 5 min and washed once with cold water (note: cell pellets can be kept frozen at -70°C after removing water). Cells are resuspended in 2 ml NIB (see original protocol) and transferred to a 15- or 50-ml conical tube containing 2 ml of glass beads (as in original protocol). Cell breakage is somewhat more rapid in 50-ml tubes, but two 15-ml tubes can be held against a Vortex mixer with one hand, so more cells can be processed at one time. In either case (15 ml or 50 ml), the tube is agitated vigorously at full speed with a Vortex mixer for 30 sec and is then placed in an ice bath for 30 sec. This operation is repeated until approximately 90% of the cells are broken (usually 1216 cycles depending on tube size, cell type and cell stage).
The supernatant is transferred to a new tube, and the glass beads are washed twice with 2 ml of NIB. The washes are combined with the original supernatant and centrifuged (cold) at 8 K rpm in a Sorvall-34 rotor for 10 min. The pellet is thoroughly resuspended in 3 ml TEN (see original protocol). Then, N-Lauroyl Sarcosine powder (ICN; a final concentration of 1.5%) and Proteinase K (a final concentration of 300 mg/ml) are added to the solution and gently mixed well. The solution is incubated at 37°C for 11.5 hrs with occasional shaking (15-20 min intervals) and then centrifuged (cold) at 5 K rpm for 5 min to pellet cell ghosts. For each ml of supernatant solution, 1.05 g of CsCl is added and dissolved by gentle mixing. The solution (3.54 ml) is transferred to an ultracentrifuge tube of appropriate volume and centrifuged at 4045 K rpm for 1824 hrs. The nuclear DNA-containing bands are collected under long wave (360 nm) UV illumination.
The DNA-containing solution is precipitated by adding 3 volumes of 75% EtOH followed by centrifugation at 12 K rpm in a Sorvall centrifuge for 20 min. The pellet is washed twice with 75% EtOH and dissolved in 150 ml of TE by incubation at 4°C for 4 hrs to overnight. One more EtOH wash to remove possible CsCl salt is recommended.
N/N 2D gel analysis
After the DNA is quantitated by gel electrophoresis with proper DNA standards (typical yield from a 200 ml culture is 3050 mg), 5 mg of the DNA (the appropriate amount for 2D gel analysis of a single copy sequence in a synchronized preparation) is digested to completion with appropriate restriction enzymes. The digested DNA is precipitated with EtOH and washed and then dissolved in 1520 ml of TE, supplemented with a loading dye, and loaded into the wells of a 0.4% (for fragment sizes of 35.5 kbp; for bigger fragments, use the modified conditions described in Hyrien and Mechali, 1992) agarose gel with a proper size marker. Since as many as 12 samples can be analyzed in one 20 X 20 cm agarose gel during the second dimension of electrophoresis, it is possible to load up to 12 samples into the same first dimension gel, so long as they are loaded in the same row of sample wells. The first dimension gel is made with 1 X TAE buffer containing 0.1 mg/ml EtBr and run at 16-17 V (for a 20 cm long gel) for 16-17 hrs or until the separation between the 1N position (the size of the non-replicating restriction fragment) and the 2N position (size of the fully replicated fragment) of the fragment of interest is 1.5-2 cm. After a picture of the gel is taken (under 360 nm UV light to minimize nicking), lanes containing DNA are cut out with a scalpel or razor blade. The gel slices should contain a region at least from 1N to 2N plus 0.5 cm on each side (in the case of 12 samples in one gel, the first dimension gel lengths should not exceed 4.5 cm each, so that four gel slices can be loaded into a single 20-cm long slot in the second-dimension gel with some room between gel slices). It is always better to use a longer gel piece containing the region of interest for future possible experiments, if there is sufficient room.
The second dimension gel, a 20 X 20 cm, 1% agarose gel, is prepared with 1 X TBE buffer supplemented with 0.5 mg/ml EtBr. Combs do not need to be used unless one wishes to generate wells for markers. In that case, we have found that a preparative comb that forms a single long preparative well with a small marker well at each end of the preparative well can be used. After the second dimension gel has solidified, troughs are cut with a sharp, clean-cutting instrument (knife, razor, scalpel or sharp spatula) to fit the first-dimension gel slices. It is important that the cuts at the lower edge of each trough (the edge through which the first dimension DNA will have to pass during second-dimension electrophoresis) be smooth and straight. If a comb was used, then the long preparative well formed by the comb should be enlarged by cutting away the agar above it so that the smooth bottom edge of the well forms the bottom edge of the resulting trough. If no comb was used, then troughs about 1.5 cm wide and as long as necessary to accommodate the first dimension gel slices are cut at the desired locations. If 12 samples are to be analyzed simultaneously, then the gel is cut horizontally at the normal well position and also at 1/3 and 2/3 of gel length. The troughs can extend across the full 20-cm width of the gel. Up to four of the first dimension gel slices can be placed end-to-end within one trough and parallel to the edge of the trough. The agarose removed during trough generation is saved for filling in around the first dimension gel slices after they have been properly placed into the trough. The saved gel pieces are thoroughly melted and the molten agar is used to fill in around the first dimension gel slices.
Second dimension gel electrophoresis is carried out in TBE (1 X, supplemented with 0.5 mg/ml EtBr) buffer at 125 V for 3 hours in the cold room.
For the next steps (gel transfer to membrane and Southern blot analysis),
see the original protocol.
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