Hybridization with Subtracted Probes

Southern and colony hybridization can with labeled subtracted material be used to assess the efficiency of subtraction and to obtain full-length cDNA clones of differentially expressed genes (Figs. 1 and 2).

1. Prepare Southern filters of the starting samples and replica filters of X cDNA libraries using standard protocols (14).

2. Label PolyAcDNA probes either by random priming or by incorporating the radioactive nucleotide by PCR using the following conditions: 1X Taq buffer, 1:100 dNTP labeling mix, 0.5 OD260/mL of the oligo used to generate the tracer, 1.2 U Taq polymerase, 5 pL 32P dCTP (final vol 100 pL). Thermal profile: 30 s at 94°C, 30 s at 60°C, 90 s at 72°C, x20 cycles.

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