Figure 3

PCR amplification of the Gm FRT cassette, PA1520 gene fragments and the overlap extension product. A. First round PCR fragments. The left panel illustrates amplification of the 1,053-bp Gm^r fragment from pPS856 which contains 24 bp (right) and 25 bp (left) overlaps with the PA1520' and 'PA1520 fragments (blue boxes). The right panel illustrates amplification of the PA1520' and 'PA1520 fragments. The 5' fragment contains 388 bp of chromosomal DNA, 25 bp overlapping the left side of the Gm^r fragment and 16 bp overlapping the GW-attB1 primer. Similarly, the 3' fragment contains 236 bp of chromosomal DNA, 24 bp overlapping the right side of the Gm^r fragment and 16 bp overlapping the GW-attB2 primer. The sequences of the gene-specific and common primers are listed in Table 1. B. Second round PCR. The purified fragments shown in panel A were used in the second round PCR illustrated in Fig. 2 to derive the indicated attB1-PA1520'-FRT-Gm-FRT-'PA1520-attB2 fragment. The entire 50 μl second round PCR reaction was subjected to agarose gel electrophoresis. The desired DNA fragment constituting the major product marked by the arrow was excised from the gel, purified and then used for the BP clonase reaction. Lanes labeled M in both panels contained Hi-Lo molecular size markers from Minnesota Molecular (Minneapolis, MN).