Generation of entry clones lacking internal BsaI sites for genes ycf4 and psbC.

Abstract

<p>(A) Three PCR fragments were amplified from <i>Arabidopsis thaliana</i> genomic (chloroplast) DNA using primers yc1–6 (gene ycf4). B, BsaI recognition sequence. The fragments were cloned in on tube into entry cloning vector pECV, resulting in entry clone pE-YCF4. Structure of the fragments for the psbC gene (not shown) is similar except that the wildtype gene contains only one internal BsaI site. (B) Restriction digest (BsaI) of minipreps from 10 white colonies for constructs pE-YCF4 and pE-PSBC. As a control, the ycf4 and psbC ORFs were amplified from genomic DNA with only the two flanking primers, and the PCR products run undigested (u, expected size 583 nt for ycf4 and 1450 nt for psbC) and digested with BsaI (d, expected sizes for ycf4: 332, 113, 54, 14, 10, expected sizes for psbC: 947, 469, 14, 10).</p