Isolation of DNA for QDFM

1. DNAs are isolated from plasmid, cosmid, P1/PAC, and BAC clones using either a commercially available purification kit or an alkaline lysis protocol (see Subheading 3.3.1. below). For the larger inserts, inserts are sized by PFGE. Digestion of DNA with a rare cutting restriction enzyme produces linear high molecular weight DNA molecules, but the alkaline lysis procedure typically provides sufficient amounts of nicked circular or randomly broken DNA suitable for QDFM (52).

2. In general, the DNA is loaded on a 1.0% low melting point agarose (Bio-Rad, Hercules, CA) gel and electrophoresed for about 15 h. To efficiently separate DNA molecules of several hundred kb, we use a PFGE system (Bio-Rad).

3.1.1. Pulsed Field Gel Electrophoresis

1. The agarose plug preparation and PFGE using a PFGE system (Bio-Rad) follow standard protocols. Typically, a diluted solution of YACs in AHC medium is plated on AHC plates, and 5 to 15 individual YAC colonies are tested to account for deletions. In most cases, the largest clone carries the least deletion(s).

2. Preparation of gel plugs containing YACs (Invitrogen; stored at -80°C): spin down cells grown in 5 mL AHC media at 30g for 6 min. Resuspend cells in 0.5 mL of 0.125 M EDTA, pH 7.8. Spin again and resuspend the cell pellet in 500 ^L of SCE. Mix with an equal volume of 1.5% low melting point (LMP) agarose preheated to 43°C. Quickly pipet up and down, and then vortex gently for 1 to 2 s to mix. Pipet into plug molds (Bio-Rad) and allow to solidify at room temperature or on ice.

3. Remove plugs from molds, incubate samples in 2 mL SCE containing 100 ^L of zymolase, and shake at 150 rpm at 30°C for 2.5 h to overnight. Replace SCE buffer with 2 mL of ES containing 100 ^L of proteinase K (20 mg/mL; Roche). Shake for 5 h to overnight at 50°C, and rinse plugs rinse 5 times with 6 mL of TE50 for 30 min each rinse. Store the plugs at 4°C until use.

4. PFGE running conditions for separation of YACs from yeast chromosomes: voltage gradient, 6 V/cm; switching interval, 79 s forward, 94 s reverse; running time, 38 h; agarose concentration, 1.0% LMP agarose; running temperature, 14°C; running buffer, 0.5X TBE.

5. PFGE running conditions for separation of full-length P1/PAC/BAC clones from debris: voltage gradient, 6 V/cm; switching interval, 2 s forward, 12 s reverse; running time, 18 h; agarose concentration, 1.0% LMP agarose; running temperature, 14°C; running buffer, 0.5X TBE (see Note 5).

6. For probe production and determination of optimal PFGE conditions: stain the gel with ethidium bromide (EB; 0.5 ^g/mL in water), cut out a gel slice containing the target DNA band, and transfer slice to a 14-mL polystyrene tube (cat. no. AS-2264, Applied Scientific). Wash slice with water for 30 min, and then wash with 1X agarose buffer for 30 min.

7. For isolation of high molecular weight DNAs: run duplicate samples on the right and left side of the gel, respectively. After a predetermined run time, cut gel in half, and stain one half with EB. Measure the migrated distance on a UV transilluminator, cut out a gel slice at the corresponding position from the unstained half, and proceed as described in step 5.

3.1.2. Recovery of High Molecular Weight DNA From LMP Agarose Gel Slices

1. The DNA is recovered from the low melting point agarose slab gel by excising the appropriate bands using a knife or razor blade. High molecular weight DNA is then isolated by P-agarose digestion of the gel slices. Equilibrate gel slice in agarose buffer.

2. Melt the gel completely by incubating it for 10 min at 85°C, and then transfer the molten agarose to a 43°C water bath.

3. Add 1 pL P-agarose for every 25 pL of molten agarose, and incubate at 43°C for 2 h.

4. Add an equal volume of 200 mM NaCl, and store the DNA samples at 4°C until use (see Note 5).

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