Alkaline lysis miniprep of plasmid DNA from E.coli

For Alkaline lysis a single bacterial colony was inoculated in 3ml of LB containing appropriate antibiotics, and grown overnight at 37°C. 1.5ml of the culture was transferred to an Eppendorf tube and the bacterial cells were pelleted by centrifugation for two minutes at 14,000 rpm in a microcentrifuge (Eppendorf).

The pellet was resuspended in 500 ml of STE (100mM NaCl, 10mM Tris.Cl pH 8.0, 1mM EDTA) and re-pelleted by centrifugation. The pellet was resuspended in 100 m l ice-cold Solution I (25mM Tris.Cl, 50mM EDTA, pH8.0, 4mg/ml lysozyme) by vortexing. 200 m l of freshly prepared solution II (0.2M NaOH, 1% SDS) was added, and mixed by slowly inverting the tube five times. Following a 5 minute incubation at room temperature 150 m l of ice-cold solution III (3M/5M KOAc, pH 4.8) was added and mixed by vortex. Samples were incubated on ice for five minutes, then centrifuged 14000 rpm (Eppendorf microcentrifuge) to remove cell debris. The supernatant was transferred to an Eppendorf tube, and DNA was ethanol precipitated. The DNA pellet was dissolved in 50 m l TE pH 8.0 or dH 20.

Phenol/chloroform extraction

To remove contaminating proteins, Phenol/chloroform solution (24 Phenol: 24 Chloroform: 1 Isoamyl alcohol (equilibrated with Tris.Cl pH 8.0; Biogene ltd.)) was added at an equal volume to the DNA samples. Samples were mixed by vortexing, then centrifuged to separate different phases. The aqueous, DNA containing phase was transferred to a fresh Eppendorf tube and DNA precipitated.

DNA precipitation

DNA was precipitated in either ethanol or isopropanol. For isopropanol precipitation 0.7 volumes of isopropanol was added to the DNA sample, and incubated at room temperature for 20 minutes. For ethanol precipitation, one-tenth volume 3M NaOAc (pH 5.2) and 2 volumes of 100% ethanol were added to the DNA sample. Followed by a 20 minute incubation at –20°C. In each case, DNA was pelleted by centrifugation at 14,000 rpm in a microcentrifuge (Eppendorf). The pellet was washed in 70% ethanol, re-pelleted and centrifuged.