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Figure 2 | BMC Genetics

Figure 2

From: Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays

Figure 2

Southern analysis of AH-4 cell line and determination of target site copy number (A): High molecular weight DNA from the AH-4 cell line (5 μg per lane) was digested separately with Bam HI (Lane B), Eco RI (Lane E), Nco I (Lane N) or Sph I (Lane S) and fractionated on 0.75% agarose alongside undigested DNA (Lane U) and untransformed (control) Mos20 DNA (Lane C). The DNA was transferred to a nitrocellulose membrane and hybridised overnight at 42°C with the hygromycin resistance gene. The membrane was washed at high stringency (0.1 × SSC; 0.1 % SDS; 60°C) and exposed to X-ray film against an intensifying screen for 5 hours at -70°C. (B): The copy number of pACT-HYG target sites within the AH-4 cell line was determined by quantitative dot-blotting. The upper panel shows hybridisation of the hygromycin resistance gene to serial dilutions (1000 to 0.01 pg; left hand scale) of supercoiled pACT-HYG (Lane 1) and serial dilutions (500 to 0.05 ng; right hand scale) of high molecular weight DNA from AH-4 cells (Lane 2). The membrane was hybridised overnight at 42°C, washed at high stringency (0.1 × SSC; 0.1 % SDS; 60°C) and exposed to a phosphor imaging screen for 2 hours. This was scanned on a Molecular Dynamics Storm 860 phosphorimager with hybridisation intensity returned as a linear scale volume report. The lower panel shows signal intensity (average volume report) from either 1.2 pg plasmid DNA or 2.5 ng AH-4 DNA.

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