2.1. The RED Method
A standard phenol/chloroform extraction method or QIAmp Blood Kit (Qiagen Inc., Chatsworth, CA) can be used for DNA preparation. Dissolve DNA in 5 mM Tris buffer, pH 9.0, with final concentration of 0.2 ^g/^L.
Oligonucleotides must be 5'phosphorylated. The denaturation/ligation cycling conditions described in this chapter are optimized for use with a (CTG)10 oligonucleotide. Please see Note 1 if other oligonucleotides are used.
1. Ampligase (100 U/|L) (Epicentre Technologies, Madison, WI, USA) with supplied buffer.
2. Terminal deoxynucleotide transferase (TdT) (Amersham, Little Chalfont, UK) with supplied buffer for end-labeling of hybridization probes.
3. Isotope a32P-dATPs (6000Ci/mmol) (NEG 012Z, NEN DuPont Medical, Wilmington, DE, USA).
4. Rapid Hybe buffer (Amersham).
5. 6% denaturing polyacrylamide/6M Urea gel solution and supplied buffer (National Diagnostics, Atlanta, GA, USA).
6. 10X TBE: 0.89 M Tris-boric acid, 20 mM Na2EDTA.
10. 20% sodium dodecyl sulfate (SDS).
11. Formamide gel loading dye: 100% formamide, 0.1% xylene cyanol, 0.1% bromophe-nol blue.
We have been using the GeneAmp PCR System 9600 (Perkin Elmer Cetus, Norwalk, CT, USA), and the PTC-225 Peltier Thermal Cycler (MJ Research, Watertown, MA, USA), but any thermocycler with a heated lid should work. In addition, Whatman 3mm filter paper, Hybond N+ membrane (Amersham) or any similar membrane, DuPont reflection, NEF 495 X-ray film, and intensifying screen or a similar product is needed.
0.2-mL microtube strips and caps are used for the RED reactions (CLP products, San Diego, CA, USA).
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