A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles
Figure 1
Elevated temperatures and osmolytes increase the efficiency of plasmid DNA extraction by NIDs.
Extractions of either pUC19 (lanes 1–8 and 10–15) or pCYPAC3 plasmids (lane 9) were carried out. Transformed DH5α cells were grown in 1.5 ml LB cultures and resuspended in 150 µl 50 mM Tris pH 8, 10 mM EDTA with or without co-solutes. 500 µg/ml lysozyme was also added, and the cells were incubated as specified below. Salt concentrations were adjusted to 0.5 M NaCl in all extracts, except the ones loaded in lanes 10 and 12–15. Extracts were cleared by centrifugation, and precipitated with 150 µl isopropanol. DNA pellets were dissolved in 40 µl TE buffer, 40 µg/ml RNase A. 10 µl aliquots of the solutions containing either the pCYPAC3 (lane 9) or pUC19 plasmids (all other lanes) were loaded on the gel. Exposure times and temperatures of extraction are shown above the lanes. For lanes 1–9, extraction with the indicated NID was done in the absence of co-solutes. For lanes 10–15, the included co-solute is indicated. Lane 1: 0.5% IGEPAL CA-630. Lane 2: 0.5% TX-100. Lane 3: 0.5% IGEPAL CA-720. Lane 4: 0.5% Tween-80. Lane 5: 0.5% Tween-20. Lane 6: 2% IGEPAL CA-720. Lane 7: 4% IGEPAL CA-720. Lane 8: 0.5% Tween 80. Lane 9: 0.5% Tween 80. Lane 10: 0.5% Tween 20/0.5 M KCl. Lane 11: 0.5% Tween 20/22.5% sucrose. Lane 12: 0.5% IGEPAL CA-630/0.5 M NH4Cl. Lane 13: 0.5% TX-100/0.5 M NH4Cl. Lane 14: 0.5% TX-100/0.5 M NaCl. Lane 15: 0.5% TX-100/0.5 M NaAc.