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. 2007 Oct;19(10):3080–3089. doi: 10.1105/tpc.107.054049

Figure 2.

Figure 2.

DNA Gel Blot Analysis of P. patens RAD51 Transformants.

(A) Genomic DNA of transformants obtained by transformation of the wild type with pKOrad51A digested with BamHI, separated by agarose gel electrophoresis, blotted, and the blot hybridized with the probe shown in Figure 1A. Pp RAD51A is located on a 3.16-kb BamHI fragment that is split in fragments of 1.16 and 2.2 kb after gene replacement. Lane 1, transformant 2/1-4; lane 2, transformant 2/1-5; lane 3, transformant 2/1-6; lane 4, transformant 2/1-7, lane 5; the wild type; lane 6, transformant 2/1-9 (Pp rad51A).

(B) Genomic DNA of transformants obtained with pKOrad51B in the wild type (transformant names starting with 1) and the Pp rad51A knockout line (transformant names starting with 7) was digested with HindIII, separated by agarose gel electrophoresis, blotted, and the blot hybridized with two different probes. Probe 1 covers the entire gene but mainly detects a 1.43-kb fragment of Pp rad51A in the wild type that shifts to 5.11 bp after replacement. Probe 2 corresponds to the region of Pp RAD51B that is deleted from the genome after gene replacement. Both probes cross-hybridize with Pp RAD51A and detect a 2.31-kb fragment in the wild type that is lost in Pp rad51A knockout lines. Lane 1, transformant 7/2 (Pp rad51AB); lane 2, 1/3 (Pp rad51B); lane 3, 1/7; lane 4, 1/5; lane 5, 1/1 (Pp rad51B-2); lane 6, 1/2; lane 7, 7/1; lane 8, 1/4; lane 9, 7/4; lane 10, 1/6; lane 11, 1/8; lane 12, Pp rad51A; lane 13, the wild type. Closed arrows indicate bands obtained by cross-hybridization with vector sequences. The open arrow indicates a vector cross-hybridizing band, except for lanes 3 and 6 in which the signal is specific for Pp RAD51B sequences.