Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes.
Plasmid DNA released from bacteria by boiling in the presence of lysozyme and Triton x-100 and without further purification can be sequenced by the dideoxy method using T7 DNA polymerase, when conditions during alkali denaturation and subsequent ethanol precipitation are adjusted to remove contaminants. The samples remain in the same microcentrifuge tubes from the harvesting of the bacteria until the splitting of the sample into four aliquots for the termination reactions. Less background label is observed with end-labelled primers (radioactivity or fluorescence), but even when radioactive nucleotides are incorporated during the sequencing reactions, 250 bases or more can be read from template prepared from 1.5 ml bacterial culture. The DNA can also be cut by restriction enzymes; the purification procedure described thus provides the rapid preparation of plasmids for a variety of purposes.
Kristensen
T
T
Laboratory for nucleic acid sequencing, Biotechnology Centre of Oslo.
Larsen
F
F
Engen
A
A
Solheim
J
J
Prydz
H
H
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005453:Fluorescence; D005821:Genetic Techniques; D006801:Humans; D009691:Nucleic Acid Denaturation; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000235:genetics; D010957:Plasmids
D004247:DNA; D007862:Phosphatidylcholine-Sterol O-Acyltransferase