Plasmid preparation


Plasmid preparation
Plasmid miniprep. 0.8% agarose gel ethidium bromide-stained.

A plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:

  • Growth of the bacterial culture
  • Harvesting and lysis of the bacteria
  • Purification of plasmid DNA

Contents

Growth of the bacterial culture

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have been transformed and isolated. Virtually all plasmid vectors in common use encode one or more antibiotic resistance genes as a selectable marker (Ex :kanamycin,Ampicillin), which allows bacteria that have been successfully transformed to multiply uninhibited. Bacterials are grown under favourable conditions.

Harvesting and lysis of the bacteria

When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.

Preparations by size

Kits are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.

Minipreparation

Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. It is based on the alkaline lysis method invented by the researchers Birnboim and Doly in 1979. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep". Minipreps are used in the process of molecular cloning to analyze bacterial clones. A typical plasmid DNA yield of a miniprep is 20 to 30 µg depending on the cell strain.

Miniprep Protocols

http://www.protocol-online.org/prot/Molecular_Biology/Plasmid/Miniprep/

Midipreparation

The starting E. coli culture volume is 15-25 ml of LB broth and the expected DNA yield is 100-350 µg.

Maxipreparation

The starting E. coli culture volume is 100-200 ml of LB broth and the expected DNA yield is 500-850 µg.

Megapreparation

The starting E. coli culture volume is 500 ml – 2.5 L of LB broth and the expected DNA yield is 1.5-2.5 mg.

Gigapreparation

The starting E. coli culture volume is 2.5-5 L of LB broth and the expected DNA yield is 7.5–10 mg.

Purification of plasmid DNA

Addition of phenol/chloroform can dissolve and denature proteins, like DNase. This is especially important if the plasmids are to be used for enzyme digestion. Otherwise, smearing may occur in enzyme restricted form of plasmid DNA.

References

Birnboim HC, Doly J (November 1979). "A rapid alkaline extraction procedure for screening recombinant plasmid DNA". Nucleic Acids Res. 7 (6): 1513–23. doi:10.1093/nar/7.6.1513. PMC 342324. PMID 388356. http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=388356. 



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