The figure below shows restriction digestion of Pseudomonas aeruginosa DNA and the plasmid pMS150A with BamHI, and Southern hybridization of the blotted restriction fragments with a biotin-labeled probe that contains the exotoxin A gene.

Lane 1- biotin labeled l Hind III molecular weight markers

Lane 2- Bam HI digest of Pseudomonas DNA

Lane 3- Bam HI digest of pMS150A (positive control plasmid containing the exotoxin A gene)

Lane 4- l Hind III molecular weight markers (unlabeled)

The probe hybridizes with several fragments in Pseudomonas genomic DNA (lane 2) and two fragments of the plasmid (lane 3). The 1.5 kb hybridizing band (arrow) contains most of the exotoxin gene. Two of the additional bands in lane 2 represent flanking restriction fragments in the Pseudomonas chromosome that contain exotoxin sequences; similarly, the one additional hybridizing band in the plasmid contains some of the exotoxin gene. The addition 3-4 bands in the Pseudomonas lane represent partial digest fragments (the other Pseudomonas strain used in the class gave complete digestion, with hybridization to only three fragments).

Exercise:

Prepare a standard curve using the biotin standards in lane 1 and calculate the size of the hybridizing fragments in Pseudomonas DNA. The size of the l Hind III markers (in kb) is: 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0. Compare results with the other Pseudomonas strain that gave only three hybridizing fragments.