REMOVING CONCANTAMERS IN NUCLINE

For high-sensitivity (low mRNA copy number) Nucline constructs, the following cloning strategy is recommended:

1) When synthesizing the Nucline sense oligo to insert into the plasmid cassette, include "sticky" overhangs directed to a restriction site in the polylinker.

2) Also, synthesize a unique restriction site upstream of the sense oligomer just before the "sticky" overhang.

3) Phosphorylate and ligate the oligomer normally into the plasmid at the polylinker site that is upstream of the effector gene.

4) Re-cut the plasmid at the unique restriction site. The concantamers will be excised, leaving only one copy of the sense oligomer.

5) Religate, transform, purify and run-off the RNA as usual. Bind RNA to the antisense molecules. Each Nucline molecule should contain only one sense and only one antisense molecule.