Plasmid DNA minipreps
To facilitate plasmid DNA minipreps a single Agrobacterium colony should be inoculated in 5ml YT containing appropriate antibiotics, and grown at 30°C for 24-36 hours. 3ml of cells are then pelleted by centrifugation, and resuspended in 100 ml GTE buffer (25mM Tris.Cl, 10mM EDTA, 50mM Glucose, pH 8.0, 4mg/ml lysozyme). 200 m l of freshly prepared solution II (0.2M NaOH, 1% SDS) is added, and mixed by slowly inverting the tube five times.
Following a 5 minute incubation at room temperature 30 ml of alkaline phenol (6 ml 10N NaOH in 1ml phenol:chloroform:isoamyl alcohol (25:24:1; v:v:v)) is added and mixed by vortex . 150 ml of ice-cold solution III (3M/5M KOAc, pH 4.8) is added, then shook by hand. Samples are incubated at –20°C for 20 minutes, and then centrifuged at 14000 rpm (Eppendorf microcentrifuge) to remove cell debris. The supernatant is transferred to an Eppendorf tube, and DNA ethanol precipitated. The DNA pellet is dissolved in 10 ml TE pH8.0 or dH2O. To check the plasmid, 1 ml of DNA solution should be transformed into E.coli, from which DNA is extracted, and analysed by restriction enzyme digest or by sequencing.