Plasmid Extraction

Summary

Purification of plasmid DNA from bacteria

Principle

Under strong alkaline conditions, plasmid DNA and genomic DNA are simultaneously released from the cell and denatured. Under pH-neutral conditions and the presence of high salt concentration, plasmid DNA will be rapidly denatured and remain soluble, with insoluble mesh structure formed by cross-linking between chromosomal DNAs. Under the action of descaling agent SDS, the chromosomal DNA combines with denatured proteins and cellular debris to form a precipitate, which is removed by a high heart and then extracted with phenol and chloroform to further purify the plasmid DNA, which can be purified by isopropanol or ethanol It can be purified by precipitation with isopropanol or ethanol.

Appliance

Plasmid Extraction

Operation method

Alkaline lysis (plasmid extraction)

Materials and Instruments

Solution I 50mmol/L glucose 25mmol/L Tris.Cl(pH8.0) 10mmol/LEDTA(pH8.0)

Move

(i) growth of bacterial cultures; (ii) harvesting and lysis of bacteria; (iii) purification of plasmid DNA

Caveat

Solution I can be prepared in batches of approximately 100 ml per vial, steam sterilized under high pressure at 10 lbf/in2 (6.895 x 104 Pa) for 15 minutes, and stored at 4 °C. To ensure that the bacterial precipitate is completely dispersed in Solution I, the bottom of two microcentrifuge tubes are shaken by touching each other to rapidly disperse the precipitate.

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