Plasmids alkaline lysis protocol

Miniprep the plasmid DNA from 1.5 mL of bactenal culture using a standard alkaline-lysis protocol (see Note 17). 

Generally, the volume of DNA should not exceed 10% of the volume of competent cells added. If it does, then lOX LiAc/TE should be added so that the transformation reaction is maintained in a IX LiAc/TE buffer. We have found that for most routine experiments, crude miniprep-DNA prepared with standard alkaline lysis protocols works well in this transformation procedure However, if the highest efficiencies are required, one should use more purified plasmid DNAs preparations (e g, cesium banded or Qiagen columns)... 

Fig. 1. Specific identification off. coli containing plasmid pBR322. Approximately 225 colonics, consisting of a 10 1 mixture of plasmid-frec and plasmid-containing cells, was grown on a nitrocellulose filter. The filterwas subjected to the lysis protocol described here, followed by a hybridization with biotinylated pBR322. Sites of positive hybridization were detected by means of streptavidin and alkaline phosphatase. The dark sites correspond to colonies harboring pBR322. Plasmid-free cells give the faint signals present at numerous sites on the filter.

M in ammonium acetate greatly improves template quality. Such a step should be examined as an amendment to any protocol that yields suboptimal template. The following protocol is a modified alkaline lysis protocol2 that in our experience routinely yields plasmid of reasonable template quality. All centrifugation steps are performed in an Eppendorf microcentrifuge (e.g., Model 5414) unless otherwise specified. The reader is referred to Sambrook et al.2 for media formulations. 

A protocol for in vitro transcription used in our laboratory is given below. Rapid plasmid minipreparations made by alkaline lysis without RNase treatment are usually sufficient to prepare DNA templates. DNA samples linearized by restriction digestion are extracted with chloroform, and precipitated with ethanol, and 0.2 to 0.5 /ig of DNA dissolved in 1 pil of distilled water is used per assay. Contamination with RNases must be avoided during the following steps. The reaction mixture for eight reactions is prepared at room temperature to avoid precipitates. 

IV A Minipreparation of mitochondria. According to Tamura and Aotsuka 1988. This alkaline lysis procedure is analogous to that used for the preparation of plasmid, covalently closed, DNA (Bimboim and Doly 1979). A similar protocol has been described for human tissues by Welter et al. 1989. 

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