School of Engineering and Science - Jacobs University

Material and Methods
4.1.3 Other bacteria
Cells of D. chrysanthemi, X. campestris and S. meliloti were grown for 1-3 days
at 28°C on LB or KB agar plates. Liquid cultures (20-250 ml) of
D. chrysanthemi were grown in Erlenmeyer flask at 28°C by shaking at 280rpm.
4.1.4 Determination of cell density
Cell density of bacterial suspensions or liquid culture was determined by
photometric measurement. The optical density (OD) of the sample was
determined at λ = 600nm. Samples with an OD above 0.6 were diluted in the
appropriate medium. It is assumed that an OD 600nm of one correlates to a cell
density of 10 9 (Miller, 1992).
4.1.5 Storage of bacterial strains
Fresh grow bacterial colonies or lawns from agar plates were resuspended in
15% sterile glycerol solution and stored at -80°C.
4.2 Molecular biology methods
4.2.1 Plasmid DNA isolation
4.2.1.1 Isolation of plasmid DNA by alkaline lysis
Small amounts of plasmid DNA were isolated by the 1-2-3 protocol based on
alkaline lysis (Sambrook et al., 1989).1-1.5 ml of E. coli overnight culture was
harvested by centrifugation at 13,000 rpm for 2-5 min. The pellet was
resuspended in 150 µl of pre-cooled buffer P1 (100 µg/ml RNase A, 50 mM
Tris/HCl, 10 mM EDTA-Na at a pH of 8.0). Alternative, cells from an overnight
LB-plate were resuspended in 150 µl of pre-cooled buffer P1. Subsequently,
the cells were lysed upon addition of 150 µl of buffer P2 (200 mM NaOH,
1% SDS) for 5 min, and the lysate was neutralized by addition of 150 µl of precooled
3 M potassium acetate solution (pH 5.5). This fast neutralisation led to
the renaturation of small plasmid DNA. Larger molecules as proteins and
chromosomal DNA, and cellular debris stay denaturated. Following incubation
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