Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Preparation of Single-Stranded Bacteriophage M13 DNA by Precipitation with Polyethylene Glycol

(Protocol summary only for purposes of this preview site)

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected bacteria into the surrounding medium. Several methods are available to purify the polymorphic filamentous particles. In this protocol, adapted from Sanger et al. (1980) and Messing (1983), the particles are concentrated by precipitation with polyethylene glycol (PEG) in the presence of high salt. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. The resulting preparation is pure enough to be used as a template for DNA sequencing. A yield of 510 g of single-stranded DNA/mL of infected cells may be expected from recombinant bacteriophages bearing inserts of 3001000 nucleotides.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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