Amplification of cDNA and Electrophoretic Analysis

1. Oligonucleotides. A various number of synthetic oligonucleotides will be required depending on the method of choice. These will be indicated in the methods sections where appropriate. The quality of the primers is of great importance, and purification is strongly recommended (see Note 4) for a brief protocol on how to purify synthetic oligonucleotides by using denaturing acrylamide gels).

2. 10X Second strand buffer;100 mM Tris-HCl, pH 8.3 at 37°C, 20 mM MgCl2, 250 mM KCl.

3. a32P-dCTP (3000 Ci/mmol, 10 mCi/mL) (see Note 5).

4. Recombinant AmpliTaq DNA polymerase (Perkin Elmer, Vaterstetten, Germany).

5. 5 M Ammonium acetate (see Note 6).

6. 10 mg/mL Oyster glycogen.

7. Urea (Boehringer Mannheim).

8. A 5% (w/v) solution of acrylamide-methylen bis-acrylamide (29:1) (Boehringer Mannheim) in 1X TBE, 50% urea.

9. Formamide loading dye: 95% deionized formamide (Gibco-BRL, Gaithersburg, MD) (v/v), 20 mM EDTA, 0.05% (w/v) bromophenol blue, 0.05% (w/v), xylene cyanol (w/v).

10. Talcum baby powder.

11. Single-sided emulsioned autoradiographic film (Kodak BioMax, Rochester, NY).

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