To isolate DNA in the size range of 150200 kb

a. Transfer the pooled aqueous phases to a dialysis bag. Close the top of the bag with a dialysis tubing clip, allowing room in the bag for the sample volume to increase 1.5-2-fold during dialysis.

b. Dialyze the solution at 4°C against 4 liters of dialysis buffer 6-1. Change the buffer three times at intervals of >6 hours.

Because of the high viscosity of the DNA solution, dialysis generally takes >24 hours to complete. To isolate DNA that has an average size of 100-150 kb a. After the third extraction with phenol, transfer the pooled aqueous phases to a fresh centrifuge tube and add 0.2 volume of 10 M ammonium acetate. Add 2 volumes of ethanol at room temperature and swirl the tube until the solution is thoroughly mixed.

b. The DNA immediately forms a precipitate. Remove the precipitate in one piece from the ethanolic solution with a Shepherd's crook (a Pasteur pipette whose end has been sealed and shaped into a U; please see Steps 5-7 of Chapter 3, Protocol 6). Contaminating oligonucleotides remain in the ethanolic phase.

c. If the DNA precipitate becomes fragmented, abandon the Shepherd's crook and collect the precipitate by centrifugation at 5000g (6500 rpm in a Sorvall SS-34 rotor) for 5 minutes at room temperature.

d. Wash the DNA precipitate twice with 70% ethanol, and collect the DNA by centrifugation as described in Step c.

e. Remove as much of the 70% ethanol as possible, using an aspirator. Store the pellet of DNA in an open tube at room temperature until the last visible traces of ethanol have evaporated.

Do not allow the pellet of DNA to dry completely; desiccated DNA is very difficult to dissolve.

f. Add 1 ml of TE (pH 8.0) for each 0.1 ml of cells (Step 1). Place the tube on a rocking platform and gently rock the solution for 12-24 hours at 4°C until the DNA has completely dissolved. Store the DNA solution at 4°C.

10. Measure the concentration of the DNA by absorbance at 260 nm or by fluorometry.

11. Analyze the quality of the preparation of high-molecular-weight DNA by pulsed-field gel electrophoresis ( Chapter 5, Protocol 17 or Chapter 5, Protocol 18 ) or by electrophoresis through a conventional 0.6% agarose gel ( Chapter 5, Protocol 1 ). Use unit-length and/or linear concatemers of X DNA as markers.

A method to generate linear concatemers of A, DNA is described in Chapter 5, Protocol 16 . Do not be concerned if some of the DNA remains in the well, since DNA molecules >250 kb have difficulty entering the gel. This problem can usually be solved by embedding the DNA in a small amount of melted agarose (at 55°C) and transferring the molten solution to the well of a preformed agarose gel.

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