Harvesting and Lysis of the Culture

Bacteria are recovered by centrifugation and lysed by any one of a large number of methods, including treatment with nonionic or ionic detergents, organic solvents, alkali, and heat. The choice among these methods is dictated by three factors: the size of the plasmid, the strain of E. coli, and the technique used subsequently to purify the plasmid DNA. Although it is impractical to give precise conditions for all possible combinations of plasmid and host, the following general guidelines can be used to choose a method that will give satisfactory results.

Large Plasmids (>15 kb in Size) Must Be Handled with Care

Plasmids >15 kb in size are susceptible to damage during both cell lysis and subsequent handling. Gentle lysis is best accomplished by suspending the bacteria in an isosmotic solution of sucrose and treating them with lysozyme and EDTA (ethylenediaminetetraacetic acid), which removes much of the cell wall. The resulting spheroplasts are lysed by adding an anionic detergent such as SDS. For methods for tender handling of large DNAs, please see the information panel on MINIMIZING DAMAGE TO DNA MOLECULES in Chapter 2.

Smaller Plasmids (<15 kb in Size) Are More Durable

When handling smaller plasmids, more severe methods of lysis can be used, and no special care need be taken to minimize shearing forces. Typically, bacterial suspensions are exposed to deter-

TABLE 1-3 Plasmid Growth and Replication

Stringent

Replicón (Example) Copy Number or Relaxed Comments

TABLE 1-3 Plasmid Growth and Replication

Modifica pMBl (pUC)

several hundred

relaxed

pUC plasmids contain a modified pMBl replicon and replicate to a very high copy number. Further amplification of the copy number by addition of chloramphenicol to the growing bacterial culture is unnecessary; instead, the culture should be grown to late log phase with vigorous shaking.

col Hi (pBR322)

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