Isolation of DNA from bacteria

The isolation of DNA from bacteria is a relatively simple

  process. The organism to be used should be grown in a favorable medium

 at an optimal temperature and should be harvested in a late log to early

 stationary phase for maximum yield. The genomic DNA isolation needs to

 separate total DNA from RNA, protein, lipid, etc. Initially, the cell

 membranes must be disrupted in order to release the DNA in the extraction

 buffer. SDS (sodium dodecyl sulphate) is used to disrupt the cell membrane

 RNA can be removed by treatment with ribonuclease (RNase) that has

 been heat-treated to inactivate any DNase contaminants; RNase is

 relatively stable to heat as a result of its disulphide bonds, which ensure

 rapid renaturation of the molecule on cooling. The other major

 contaminant, protein, is removed by shaking the solution gently with

 water-saturated phenol, or with a phenol/chloroform mixture, either of

  which will denature proteins but not nucleic acids

.

The deproteinised DNA

 preparation is mixed with two volumes of absolute ethanol, and the DNA

 allowed to precipitate out of solution in a freezer. After centrifugation, the

 DNA pellet is redissolved in a buffer containing EDTA to inactivate any

 DNases present. This solution can be stored at 4oC for at least a month.

 DNA solutions can be stored frozen although repeated freezing and

 thawing tends to damage long DNA molecules by shearing. The procedure

 described above is suitable for total cellular DNA. If the DNA from a specific

 organelle or viral particle is needed, it is best to isolate the organelle or

 virus before extracting its DNA, since the recovery of a particular type of

 DNA from a mixture is usually rather difficult. Where a high degree of purity

 is required DNA may be subjected to density gradient ultracentrifugation

 through caesium chloride which is particularly useful for the preparation of

 plasmid DNA