Rapid Isolation of Plasmid DNA (Miniscreen)
Procedures for a large-scale isolation arid purification of plasmid DNA are elaborate, time-consuming and require ultracentrifugation. However, there shall be occasions when one has to analyze a large number of samples for the presence of a plasmid and also do not need either to isolate large quantity of plasmid DNA or of high purity. There are several procedures that describe the isolation of small amounts of partially purified plasmid DNA from a large number of bacterial clones in a relatively short period of time. One such 'miniscreen' procedure is described below. This method does not require a large input of cells and can be carried out in small Eppendorf tubes.
The isolation procedure is based upon the release of soluble high MW DNA from disrupted cell wall and membranes, dissociation of nucleoprotein complexes by denaturation and proteolysis and the separation of DNA from other macromolecules.
» Solution A (pH 8.0)
25mM Tris-HCl 30.3mg
50mM glucose 90mg
Lysozyme 100mg (freshly added before use)
» Solution B
0.2M NaOH 0.8g
1.0% SDS 1.0g
» Solution C
3M sodium acetate (pH 4.8) 24.6g
Adjust pH with glacial acetic acid and then make up the volume
» Solution D
50mM Tris-HCl (pH 8) 0.60g
100mM sodium acetate 8.20g
1. Clewell, D B and Helinski, D R (1972) J. Bacteriol 110 1135.
2. Marko, M A, Chipperfield, R and Birnboim, H C (1982) Anal Biochem 121 382.
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