Removal of Linkers and Small cDNAs

1. Pour a column in a 1-mL disposable pipet plugged with sterile polyester wool (see Note 9).

2. Make a reservoir with a 2-mL disposable Pasteur pipet cutting the bottom off at the appropriate level and slipping over the end of the column. Now cut the very top of the pipet off, fill, and rinse with 10 mL of column buffer.

3. Resuspend the cDNA in 50 |L of column buffer, and apply to the column (see Note 10).

4. Monitor the progress of the radioactivity into the column with a Geiger counter. When it gets one-third of the way (~250 |L), start collecting two-drop fractions into microfuge tubes. Continue collecting fractions until the major 32P peak (linkers) reaches the bottom.

5. Count the all of the fractions by Cerenkov counting, and plot the results. You should see two distinct peaks.

6 . Run aliquots of alternate column fractions on a 1% agarose gel with labeled 1-kb ladder and unlabeled 1-kb ladder.

7. Dry the gel and expose to film. Pool the peaks containing cDNA from about 500 bp to the beginning of the column (see Note 11).

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