1. Add an appropriate amount of vector to the pooled cDNA and ethanol-precipitate (overnight is best), rinse carefully, and dry (see Note 12). Try to use about 10X the weight in cDNA of X arms. The use of less will result in a significant proportion of clones containing multiple inserts, although the total number of clones will increase.
2. Resuspend the cDNA/vector pellet in 5 ||L of ligation cocktail, which contains the following:
0.5 |L 10X ligation buffer;
3. Be sure the pellet is completely dissolved, and centrifuge to put everything into the bottom of the tube.
4. Ligate at 14°C overnight.
Was this article helpful?