1. Molecular biology grade agarose (Gibco-BRL, Gaithersburg, MD).
2. 10X TBE: 108 g Boric acid, 55 g Tris base, and 40 mL 0.5M EDTA, pH 8.0, to 1 L. Autoclave to sterilize.
3. 20 x 20 cm gel casting tray, running tank, and electrophoresis power pack.
4. 10 mg/mL ethidium bromide in water (caution: extremely carcinogenic).
5. 20X SSPE: 3.6 M NaCl, 200 m M Na phosphate, pH 6.8,20 mM EDTA.
6. 10X loading dye: 40% (v/v) glycerol, 10X TBE, 0.1 % (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol.
7 Nylon hybridization membrane (e.g., Amersham [UK] Hybond-N; see Note 1).
8. Capillary transfer system (see Note 2).
9. Depurination buffer: 0.25M HC1.
10. Denaturation buffer: 1.5MNaCl, 0.5MNaOH.
11. Transfer buffer: 1.5M NaCl, 0.25M NaOH.
12. 312 nm UV light transilluminator. 13 Copy number standard (see Note 3).
14. Markers (size standards; see Note 4).
15. Genomic DNA (from tail biopsy; see Chapter 34).
16. Restriction enzymes and buffers (see Chapter 50; see Note 5).
17. Labeled probe DNA (see Chapter 53).
1. Cut 10-15 pg of genomic biopsy (usually tail) DNA (Chapter 34) with an appropriate restriction endonuclease as described (Chapter 50).
2. Make an agarose gel as described (Chapter 51).
3. To the digest, add 1/10 vol of loading dye, mix well, and load the samples into the gel slots. Include appropriate standards on the gel (see Notes 3 and 4).
4. Apply a current across the gel (negative to positive, 4 V/cm) and run until the fast (bromophenol) blue has reached the end of the gel.
5. After electrophoresis, illuminate the gel using a UV light transilluminator (312 nm). Photograph the gel with a ruler placed alongside the marker DNA lane.
6. Remove unused portions of the gel with a clean scalpel blade.
7. Incubate the gel in approx 3 gel vol of depurination buffer with gentle agitation at room temperature for 30 min, or until the bromophenol blue in the loading dye turns yellow.
8. Decant the depurination buffer and replace with 3 gel vol of denaturation buffer. Incubate with gentle agitation at room temperature for 30 min.
9. Decant the denaturation buffer and replace with 3 gel vol of transfer buffer. Equilibriate the gel with gentle agitation at room temperature for 30 min.
10. Place the gel on the platform of a capillary transfer system filled with transfer buffer. A diagram of a transfer system is shown in Fig. 1. The system is made up of a platform that sits in a reservoir containing transfer buffer. A wick, made up of four thicknesses of 3MM paper is placed over the platform, soaked in transfer buffer. All air bubbles must be removed from the wick. The width and length of the platform corresponds to the size of the gel and the wick is cut to the same width. The platform and reservoir are made to the same height.
11. Cut a piece of Hybond-N (Amersham) to the same size as the gel. Wet this by floating it on distilled water then rinse in transfer buffer. Place the filter on the gel and smooth out any air bubbles.
12. Cut four pieces of 3MM paper to the same size as the gel. Soak two in transfer buffer and place over the filter. Smooth out any bubbles. Place two dry filters onto the sandwich, then a stack of dry paper towels. Place a 1-kg weight on top and allow transfer to proceed at least 12 h.
13. Disassemble the transfer system. Prior to separating the gel from the filter, the position of the gel slots can be marked. If this is done with a
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