DNA clean-up
Fragment isolation
Competent cells
Probe Labeling
Probe Detection

Plasmid isolation

Quick ("and dirty") plasmid preparation from a small number of cells (e.g. in 1.5 ml overnight bacterial cultures) is often referred to as a "miniprepping".

The general procedure involves six successive steps:

  1. Lyse the cells with a mixture of NaOH and sodium dodecyl sulfate (SDS). NaOH will denature DNA and partially hydrolyze RNA. SDS is a very powerful ionic detergent that will dissolve membranes and denature proteins.
  2. Precipitate the SDS-protein/cell debris with a high concentration of sodium or potassium acetate of low pH (4.8 - 5.2); K- or Na dodecyl sulfate is largely insoluble in water. The low pH of the acetate is important, as it neutralizes the alkaline NaOH used in the previous step and allows renaturation of the plasmid DNA. Small, covalently closed, circular plasmids, which have both strands at close proximity after denaturation, will snap back into their original hydrogen-bonded, double-stranded conformation. Denatured chromosomal DNA will be unable to renature due to its large size (complexity) and instead will form small regions of double-stranded structures, in regions of similar sequence, that will cause aggregation. Addition of acetate will cause a large clotting of most of the cellular DNA, membranes, and many assorted proteins. Most of the plasmids and cellular RNA will not clot. Gentle, but complete, mixing is important, since vigorous shaking of the mixture will break the bacterial chromosome into small fragments that will not clot and would thus contaminate the plasmid preparation.
  3. Centrifuge the lysate to pellet the clot; the plasmids should be in the supernatant.
  4. Precipitate the nucleic acids in the supernatant with ethanol or isopropanol to permit concentration of the plasmids and remove many of the RNA fragments. The precipitation is done at room temperature to reduce the precipitation of smaller RNA fragments.
  5. Wash the DNA/RNA pellet with 70% ethanol to remove salts, (this keeps the DNA and RNA insoluble), dry it and
  6. dissolve it in a small volume of a low salt buffer (or water). This buffer may contain RNase to breakdown the RNA in the miniprep.
    The final solution may contain white insolubilities, which can easily be removed by centrifugation.

The yield of (high-copy) plasmid DNA from bacterial cultures vary, but is usually between 1 and 3 μg  from 1.5 ml of overnight bacterial culture.
For reasons unknown sofar, Simple Cloning Lab clones pGTλ40 and pGTλ52 (containing the 3240 and 2752 bp Lambda inserts, resp.) always give a much lower yield.

If you would have to analyse miniprep by restriction analysis, consider making a so-called Mastermix...