Construction and amplification of CLIPs.

<p><b><b><i>A</i></b><b>.</b></b> Test of pLib2 vector self-ligation alone (lane 2) or in the presence of a 1000-fold excess of random PA DNA duplex digested with Apa I (lane 3). Excess PA does not affect the self-ligation of vector with incompatible ends (BamHI) (lanes 4 and 5). When pLib2 is digested with EcoRI, dephosphorylated with Antarctic Phosphatase (AP) and digested again with ApaI, the ligation reaction goes almost to completion with negligible amounts of vector self-ligation products formed (lanes 6 and 7). RI– EcoR I, AP– Antarctic Phosphatase. Lane 1 is linearized pLib2 plasmid and lane 8 is DNA molecular weight markers. <b><i>B</i></b><b>.</b> Typical separation profile of pLib2/Apa I + PA duplex ligation products from excess duplex and other reactants on a Sephacryl S500 column. <b><i>C</i></b><b>.</b> Example of amplification of ”recircularization” ligation (see text) with Phi 29 polymerase. Ligation reactions were split into smaller batches, amplified with Phi 29 and digested with BamHI (Lanes 3–6, respectively). Phi 29 amplification depends strongly on the quality of circular template and can fail if the yield of circular ligation product is low (Lane 6). Plasmid pLib2 was amplified under the same conditions and served as a control (Lane 2).