>> I'm looking for a sure-fire fool-proof absolutely guaranteed DNA
> prep protocol that gives nice clean sequence with the sequenase kit.
>http://sp1.berkeley.edu/The best results I have are with a three steps alcaline lysis miniprep.
1) resuspend your bacteria pellet in 200 µl lysis buffer (50mM glucose,
25mM tris HCl pH 8, 10mM EDTA, 4mg/ml lysozyme
2) at RT add 400µl of 0,2M NaOH:1% SDS
3) add 300 µl 7.5M ammonium acetate (pH 7,8 without adjustment). It is
important to use ammonium acetate and not potassium acetate.
4) maintain the tube for 10min at O°C. Add a drop of chloroform (it
helps a lot as the white precipitate do not flotate any longer) and
5) save the clear supernatant and precipitate with 540 µl isopropanol
6) centrifuge, wash the pellet and resuspend in 50µl TE + 10µg/ml RNASE
7) use 8µl for the sequence. It has always worked with classical high
copy number plasmids
Florence Cabon. CNRS Villejuif, France. cabon at infobiogen.fr