Preparation of the sequencing primers

The primer is prepared from an EcoRl digest of the RF1 DNA, isolated as described above, from M13mp2962 (Heidecker et al., 1980), and separated by polyacrylamide gel electrophoresis or RPC-5 chromatography. 

The 96-nucleotide primer can also be isolated by HPLC on RPC-5 at neutral pH (Eshaghpour and Crothers, 1978, Sanger et al., 1980). For a discussion on the application of this technique to nucleic acid fractionation see Wells et al. (1980). 

Plate out a sample of E. coli carrying pSP14 onto a YT plate containing 25 /xg/ml ampicillin and grow overnight at 37°C. Resuspend the resultant colonies into 5 ml 2YT and use this to inoculate 1 litre 2YT in a 2 litre flask. Incubate with shaking at 37°C up to a cell density of approximately 0.8 A o units add 150 mg chloramphenicol and incubate for an additional 16 h at 37°C. 

Harvest the cells and prepare a cleared lysate as previously (4.3.4.), except that the volumes are increased to give approximately 40 ml cleared lysate. Adjust to 0.1 M NaCl, 0.2% Sarkosyl, and add 4 mg pancreatic RNAase (from a 5 mg/ml stock solution in 0.1 M sodium acetate, pH 5.5, previously boiled for 10 min.). Incubate for 1 hour at 37°C. Add 1.0 ml of a 1 mg/ml solution of proteinase K (BDH Biochemicals) and continue incubation for a further hour at 55°C. 

Extract crude lysate twice with 0.5 vol. phenol (redistilled equilibrated with 0.2 M Tris-HCl, pH 7.8) plus 0.5 vol. chloroform isoamyl alcohol (24 1). Precipitate with ethanol and dissolve DNA in 15 ml of 10 mM Tris-HCl (pH. 78), 1 mM-EDTA. 

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