Nucleic Acids Research, Vol. 18, No. 18 5573
A simplified and improved method for the efficient doublestranded sequencing of mini-prep plasmid DNA W.M.Wong, D.M.Y.Au, V.M.S.Lam, J.W.O.Tam and L.Y.L.Cheng Department of Biochemistry, University of Hong Kong, Hong Kong Submitted August 6, 1990
Mini-prep of plasmid DNA followed by restriction mapping and sequencing is useful for analyzing a DNA fragment of interest. Numerous methods for fast mini-prep of DNA have been proposed, but the isolated DNA is either not pure enough for sequencing (1, 4, 6), or the procedures involved the use of enzyme inhibiting denaturating agents (2, 7), enzymes treatments (3), or multi-step DNA precipitation (5), which are time consuming and tedious. A 20 min DNA mini-prep kit is commercially available (LKB-Pharmacia), however, fine adjustment of the kit to suit different purposes is not possible. By combining alkaline lysis and salt precipitation of high MW RNA into one step, removal of low MW RNA and protein into the second step, mini-prep of plasmid DNA suitable for sequencing can be done within 20 min. Centrifuge 1.5 ml saturated O/N culture at 12000 g for 1 min to collect the bacteria. Resuspend the pellet in 100 1l buffer containing 25 mM Tris-Cl, 150 mM NaCl, 10 mM EDTA at pH 8.0, add 200 Al of 0.2 N NaOH/1 % SDS. Tap to mix and observe for lysis of the bacteria, then add 600 Ml 1.5 M potassium acetate pH 4.5/4.5 M LiCl and mix. Spin at 12000 g for 5 min to remove the precipitated cell debris, chromosomal DNA, and high molecular weight RNA. To the supematant, add 540 A1 propan-2-ol, mix and centrifuge 12000 g for 5 min. Dissolve the DNA pellet in 100 1l TE. Spin the DNA through a self-packed TE pre-equilibrated Sephacryl S-400 HR (LKB-Pharmacia) 1 ml mini column once or twice, depending on the efficiency of the mini column. The eluted DNA is now suitable for sequencing. Yield of the DNA is about 5 ,sg. To 30 A1 (about 2 1tg DNA) of the DNA solution, denature by adding 3 Id of 2 N NaOH and incubate at RT for S min. In order to minimize self-annealing of the denatured DNA which brings about the undesired cross-banding in double-stranded DNA sequencing, it is important to add 120 IL ice cold absolute ethanol prior to the addition of 15 jdl IM sodium acetate at pH5 for neutralization. Allow the DNA to precipitate at RT for 5 min, then centrifuge and dry. Add 30 ng sequencing primer to the dried DNA first, otherwise, the annealing mixture is made up according to the manufacturer's protocol of the Sequenase DNA Sequencing Kit (United States Biochemical Corp). The mixture is annealed at 37°C for 45 min. All other subsequent mixing and reactions are carried out as instructed in the supplied protocol. A sample of our result is shown in figure 1.
Figure 1. Plasmid DNA isolated by mini-prep and sequencing reaction carried out as described.
REFERENCES 1. Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience. 2. Bansal,O.B. and Das,R.H. (1989) Nucl. Acids Res. 17, 10129. 3. Grimberg,J., Maguire,S. and Belluscio,L. (1989) Nucl. Acids Res. 17, 8893. 4. He,M., Wilde,A. and Kaderbhai,M.A. (1990) Nucl. Acids Res. 18, 1660. 5. Sal,G.D., Manfioletti,G. and Schnider,C. (1988) Nucl. Acids Res. 16, 9878. 6. Sambrook,J., Fritsch,E. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 7. Serghini,M.A., Ritzenthaler,C. and Pinck,L. (1989) Nucl. Acids Res. 17, 3604.
A simplified and improved method for the efficient doublestranded sequencing of mini-prep plasmid DNA W.M.Wong, D.M.Y.Au, V.M.S.Lam, J.W.O.Tam and L.Y.L.Cheng Department of Biochemistry, University of Hong Kong, Hong Kong Submitted August 6, 1990
Mini-prep of plasmid DNA followed by restriction mapping and sequencing is useful for analyzing a DNA fragment of interest. Numerous methods for fast mini-prep of DNA have been proposed, but the isolated DNA is either not pure enough for sequencing (1, 4, 6), or the procedures involved the use of enzyme inhibiting denaturating agents (2, 7), enzymes treatments (3), or multi-step DNA precipitation (5), which are time consuming and tedious. A 20 min DNA mini-prep kit is commercially available (LKB-Pharmacia), however, fine adjustment of the kit to suit different purposes is not possible. By combining alkaline lysis and salt precipitation of high MW RNA into one step, removal of low MW RNA and protein into the second step, mini-prep of plasmid DNA suitable for sequencing can be done within 20 min. Centrifuge 1.5 ml saturated O/N culture at 12000 g for 1 min to collect the bacteria. Resuspend the pellet in 100 1l buffer containing 25 mM Tris-Cl, 150 mM NaCl, 10 mM EDTA at pH 8.0, add 200 Al of 0.2 N NaOH/1 % SDS. Tap to mix and observe for lysis of the bacteria, then add 600 Ml 1.5 M potassium acetate pH 4.5/4.5 M LiCl and mix. Spin at 12000 g for 5 min to remove the precipitated cell debris, chromosomal DNA, and high molecular weight RNA. To the supematant, add 540 A1 propan-2-ol, mix and centrifuge 12000 g for 5 min. Dissolve the DNA pellet in 100 1l TE. Spin the DNA through a self-packed TE pre-equilibrated Sephacryl S-400 HR (LKB-Pharmacia) 1 ml mini column once or twice, depending on the efficiency of the mini column. The eluted DNA is now suitable for sequencing. Yield of the DNA is about 5 ,sg. To 30 A1 (about 2 1tg DNA) of the DNA solution, denature by adding 3 Id of 2 N NaOH and incubate at RT for S min. In order to minimize self-annealing of the denatured DNA which brings about the undesired cross-banding in double-stranded DNA sequencing, it is important to add 120 IL ice cold absolute ethanol prior to the addition of 15 jdl IM sodium acetate at pH5 for neutralization. Allow the DNA to precipitate at RT for 5 min, then centrifuge and dry. Add 30 ng sequencing primer to the dried DNA first, otherwise, the annealing mixture is made up according to the manufacturer's protocol of the Sequenase DNA Sequencing Kit (United States Biochemical Corp). The mixture is annealed at 37°C for 45 min. All other subsequent mixing and reactions are carried out as instructed in the supplied protocol. A sample of our result is shown in figure 1.
Figure 1. Plasmid DNA isolated by mini-prep and sequencing reaction carried out as described.
REFERENCES 1. Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience. 2. Bansal,O.B. and Das,R.H. (1989) Nucl. Acids Res. 17, 10129. 3. Grimberg,J., Maguire,S. and Belluscio,L. (1989) Nucl. Acids Res. 17, 8893. 4. He,M., Wilde,A. and Kaderbhai,M.A. (1990) Nucl. Acids Res. 18, 1660. 5. Sal,G.D., Manfioletti,G. and Schnider,C. (1988) Nucl. Acids Res. 16, 9878. 6. Sambrook,J., Fritsch,E. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 7. Serghini,M.A., Ritzenthaler,C. and Pinck,L. (1989) Nucl. Acids Res. 17, 3604.
