Pulsefield gel electrophoresis procedures

We routinely use the CHEF-DR II or III pulse-field electrophoresis system (BioRad). The basic equipment is as described by the manufacturer. The CHEF-DR II system is an advanced pulse-field system based on the CHEF (clamped homogeneous electric fields) technique. The key to high resolution, sharp bands, straight lanes, and reproducible separations is a uniform electric field at all points of a gel, and an optimal 120 "angle of alternating pulses. The CHEF-DR II system accomplishes both of these by establishing the electric field along the contour of the hexagonal array of 24 electrodes. The basic unit of the CHEF-DR II system includes:

• Pulsewave 760 switcher

• model 200/2.0 power supply

The procedures used are as described in Protocol 1.

Protocol 1. Pulse-field gel electrophoresis procedures

Solutions

mM NaCI, pH 7.8. • deproteinizing solution: 0.1 mg/ml proteinase K, 1% sarkosyl, 50 mM EDTA, 50 mM Tris-HCI, pH 7.8.

• plug-washing buffer: 10 mM Tris-HCI, 1 mM EDTA, pH 7.8.

• 0.5 x TBE running buffer: 45 mM Tris base, 45 mM boric acid, 1 mM EDTA, pH 8.2.

A. Sample preparation in agarose blocks

Any technique for the analysis of high molecular weight DNA fragmentation is critically dependent on its ability to isolate intact DNA from control cells and prevent further degradation of DNA from apoptotic cells. The technique of embedding cells in agarose plugs was evolved to solve this problem. Large DNA fragments are so fragile that they are broken by mechanical forces during their isolation. To prevent breakage of these large DNA fragments, intact cells embedded in agarose are lysed and deproteinized. The agarose matrix protects the embedded DNA from shear forces and provides an easy way to manipulate samples. Processed agarose plug-DNA inserts are loaded directly into sample wells of agarose electrophoresis gels.

Method

1. Wash ~2 x 10® cells in phosphate-buffered saline (PBS).

2. Pellet the cells by centrifugation at 4°C for 10 min at 3000 r.p.m. (450 g).

3. Discard the supernatant and resuspend the cells with an equal volume of TEN buffer.

4. Prepare 1% low-melt preparative grade agarose (low melting point agarose) solution in TEN buffer by melting agarose in a microwave oven. Let the agarose solution cool to 50°C.

5. Add the 1% melted agarose to the cell suspension to give a final agarose concentration of 0.75%.

6. Pipette into mould chambers and allow cooling to 4°C for ~20 min.

7. The agarose plugs are removed from the mould and then incubated in deproteinizing solution at 45°C for 16 h.

8. Wash with plug-washing buffer once an hour, three times.

B. Gel casting Method

1. Place the casting stand on a levelled surface.

2. Attach a comb to the holder and adjust the height of the comb to ~2 mm above the casting stand surface.

3. Prepare the desired concentration of agarose (see Section 3.2) in 0.5 x Tris-borate electrophoresis buffer.

4. Melt the agarose in a microwave oven.

5. Pour the molten agarose into the casting stand and allow to cool for 1 h at room temperature.

6. Carefully remove the comb holder and comb.

7. Sample plugs can be added to the wells while the electrophoresis gel remains in the casting stand.

C. Loading the sample Method

1. Place the sample plugs on a smooth clean surface, and cut them to size. Samples should be less than 90% of the height of the wells.

Protocol 1. Continued

2. Fill each sample well with low-melt agarose at an agarose concentration equal to that of the gel, and allow the agarose to harden at room temperature for 15 min.

D. Running the gel

Method

1. Pour 0.5 x TBE buffer into the chamber.

2. Turn on the recirculating pump, and allow the buffer to equilibrate to the desired temperature (14°C is recommended).

3. Slide the gel on to the surface of the chamber. Check the buffer level so that the gel is covered by about 2 mm buffer.

4. Turn on the Pulsewave 760 switcher and power supply. Set the Pulse-wave 760 parameters (the initial and final pulse time, ratio, running time) and voltage. For details of the conditions for electrophoretic separations see Section 3.2.

E. Staining the gel

Method

1. After electrophoresis, place the gel into 0.5 |xg/ml ethidium bromide solution and let it stain for 20-30 min.

2. Destaining is performed in distilled water for 1-3 h.

3. The DNA can be visualized by placing the gel on a UV transilluminator (254-360 nm).

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