High-throughput purification of M13 templates for DNA sequencing.
Two variants of methods for high-throughput preparation of single-stranded M13 DNA are described. Both variants are derived from previously described chemistry and are appropriate for purification of M13 templates in 96-deep well plates. In both variants, phenol extraction is replaced by treatment with sodium iodide to disrupt phage proteins prior to ethanol precipitation of M13 DNA. In one of the variants, nonderivatized paramagnetic particles are employed to collect aggregated M13 phage particles and DNA, thereby replacing the need for centrifugation. The other variant omits the magnetic particles and utilizes a centrifuge that can accommodate the 96-deep well plates. Although the purification scheme that uses magnetic separation results in a decreased yield of M13 DNA, it is amenable to robotic automation strategies and thus will be useful for genomic sequencing projects. Performed manually, either method can easily produce 192 templates in a few hours. Although both variants produce DNA of sufficient quantity for automated fluorescent DNA sequencing, the procedure that utilizes magnetic separation provides template DNA of higher quality.
Wilson
R K
RK
Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110.
D001322:Autoanalysis; D017104:Bacteriophage M13 / Q000235:genetics*; D002498:Centrifugation; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification*; D008280:Magnetics; D008863:Microspheres; D019800:Phenol; D010636:Phenols; D011092:Polyethylene Glycols; D017422:Sequence Analysis, DNA / Q000379:methods*; D012974:Sodium Iodide; D013698:Templates, Genetic
D004279:DNA, Viral; D010636:Phenols; D019800:Phenol; D011092:Polyethylene Glycols; D012974:Sodium Iodide