Preparation of single-stranded M13 DNA templates

Recombinant DNAs with inserted Sou 3A frogment (two orientations) 

50 /xl 10 mM Tris-HCl, pH 7.9, 1 mM-EDTA are added followed by 50 /x 1 water-saturated phenol equilibrated with the above buffer. The mixture is vortexed briefly, allowed to stand for 30 min., revortexed, and centrifuged for 1 min. The upper aqueous phase is removed (about 45 /xl) into another Eppendorf tube and extracted three times with ether. Finally, 10 /xl 1 M sodium acetate pH 5.5 and 140 fil 95% ethanol are added and the mixture left at -20°C overnight. The DNA is collected by centrifuging for 10 min. and the supernatant removed as previously. The precipitate is barely visible. The pellet may be washed with 0.5 ml 95% ethanol but great care has to be taken not to lose the pellet. 

The precipitate is air-dried, dissolved in 50 /xl H20 and stored frozen at -20°C. These samples provide the single-stranded templates for sequencing. 

40 p primer (derived from 50 pg M13mp2962 RF or pHM232) (Section 4.3.7.) 

Heat to 75°C for 10 min. to kill the enzyme This gives sufficient primer for 20-30 sequencing experiments. 

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