Method

1. Cast an agarose gel of the appropriate concentration in 0.5x TBE buffer as described in Chapter 5,

Protocol 1 . Use a bubble level to ensure that the casting tray is completely flat on the laboratory bench. Allow the gel to harden for 1 hour at room temperature.

2. Place the agarose gel in the CHEF apparatus, add enough 0.5x TBE to just cover the gel, and cool the remaining buffer to 14°C.

3. Prepare agarose plugs containing the DNA of interest (please see Chapter 5, Protocol 13 for preparation of mammalian DNA or Chapter 5, Protocol 14 for preparation of yeast DNA embedded in plugs) and carry out digestion with restriction enzymes as described in Chapter 5, Protocol 15 . Prepare and embed the appropriate DNA size standards as described in Chapter 5, Protocol 14 or Chapter 5, Protocol 16 .

4. Gently place the plugs in individual microfuge tubes and add 200 pl of 0.5x TBE to each. Incubate the plugs for 15 minutes at room temperature.

5. Embed the digested and rinsed DNA plugs in individual wells of the gel. Seal the plugs in the wells with the same solution of molten agarose used to pour the gel.

6. Allow the sealed plugs to harden in the gel for approx. 5 minutes, and then add additional 0.5x TBE buffer (previously cooled to 14°C in Step 2) to the apparatus to cover the agarose gel completely.

7. Start the buffer circulating and begin the electrophoresis run using power supply settings as described in the table below.

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