Linker Ligation see Note

1. Resuspend cDNA in 27 pL ddH2O. An example reaction is the following:

27 pL cDNA;

5 pL 10X ligase buffer;

6 pL 32P-labeled linkers—0.8 pg;

3 pL unlabeled linkers—3 pg;

2 pL T4 RNA ligase—12-18 U (see Note 6). Incubate for 1 h at room temperature then 14°C overnight.

2. Heat-kill the enzyme at 80°C for 15 min, and then cool on ice.

3. Remove 1 pL of the ligation mix to test for ligation (see Note 7).

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