A small-scale plasmid preparation yielding DNA suitable for double-stranded sequencing and in vitro transcription.
A small-scale plasmid preparation is described that is useful for a variety of procedures from double-stranded sequencing to in vitro transcription. No specialized equipment or reagents are required. The preparation of plasmid DNA does not require the use of RNase; instead the larger RNAs are precipitated with 2.5 M ammonium acetate. The resulting plasmid DNA is used routinely for double-stranded sequencing with the Klenow fragment of DNA polymerase and has been used for generating deletions with exonuclease III. In addition, the plasmid DNA has been used to generate transcripts with T7 RNA polymerase that translate well in reticulocyte lysate.
Johnson
K R
KR
Department of Biology, University of Toledo, Ohio 43606.
D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D009710:Nucleotide Mapping; D010957:Plasmids; D012313:RNA / Q000302:isolation & purification; D014158:Transcription, Genetic; D014764:Viral Proteins
D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases