Use of the Subtracted cDNA

1. Construction of a subtracted cDNA library. Begin with Subheading 3.4.2., step 2—second-strand synthesis.

2. Preparation of a subtracted cDNA probe. Divide the cDNA into three portions. Reagents for random priming are prepared as follows: 1 M HEPES, pH 6.6, DTM (0.1 mM each of dGTP, dTTP in 0.25 M Tris-HCl, pH 8.0, 0.025 mM MgCl2, 0.05 mM BME), OL (1 mM Tris-HCl, pH 7.5, 1 mM EDTA containing 90 OD U of random hexanucleotides [Pharmacia]/mL), LS (1 M HEPES:DTM:0L [25:25:7] [v/v]) (15) (see Note 18).

3. The reaction mix contains 11.4 |L solution LS, 1 |L BSA (Sigma) (10 mg/mL), one-third of the subtracted cDNA fragment and H20 to 37.5 |L.

4. Boil this reaction for 5 min, and then snap-cool on ice.

5. Add 5 pL each of a-[32-P]dATP and dCTP (3000 Ci/mM) and 2.5 U of the Klenow fragment of DNA polymerase I (Pharmacia).

6. Incubate for 2 h to overnight at room temperature.

7. Remove the unincorporated nucleotides by Sephadex G-50 spun-column chro-matography as above.

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