Plasmid preparation
The plasmid (also plasmid isolation ) is a molecular biological method for the isolation of plasmid - DNA . The plasmid preparation is a variant of the DNA extraction .
application
The use of plasmids makes it possible to reproduce genetic material in bacteria or to transfer certain genes to other organisms. The yield of plasmids depends, among other things, on the bacterial strain, the culture medium used , the origin of replication of the plasmid (so-called high copy plasmids or low copy plasmids ) and the culture conditions ( shaker up to 200 rotations per minute, shape of the culture bottle such as e.g. Baffle piston , temperature). The miniprep is available for isolating small amounts of plasmids (up to 25 µg), the maxiprep is suitable for larger amounts up to 0.5 mg, the megaprep up to 2 mg and the gigaprep up to 10 mg. The plasmid preparation is used to isolate preparative plasmids from transformed bacteria, mostly Escherichia coli .
Alkaline lysis
There are several options for plasmid preparation, one of which is " alkaline lysis ". The bacteria with the plasmid are centrifuged at approx. 5000 xg and the pellet is then resuspended in a buffer . For the degradation of interfering RNA , the solution usually also contains an RNase . By adding a solution of SDS and sodium hydroxide , the cells are lysed by the detergent and the alkaline saponification of the lipids .
By adding NaOH to the cell extract, the pH value is shifted far into the alkaline range. As a result, the hydrogen bonds between the complementary DNA strands of both the chromosomal and the plasmid DNA are broken, the plasmid DNA being able to completely renature due to its conformation. The chromosomal DNA, which was broken up by the individual preparation steps, cannot renature after neutralization of the pH value with potassium acetate and glacial acetic acid, DNA double strands with only short complementary areas are formed and the undirected connection of many DNA single strands leads to formation a tangled mass of DNA. This can be centrifuged off relatively easily. During this centrifugation step, cell membrane and cell wall components as well as proteins are deposited as pellets. The plasmid DNA is located in the supernatant after centrifugation.
The dissolved plasmid DNA can be subjected to additional purification, e.g. B. phenol-chloroform extraction or adsorption on silica gel . The DNA can then be precipitated with an alcohol such as ethanol or isopropanol . The alcohol removes the hydration shell from the DNA . The precipitated DNA is then washed with 70% ethanol and is then available for further processing. To measure the concentration in the photometer , the DNA can be dissolved again in water or TE buffer (pH 7.5), for example.
Koch lysis
Another digestion method is e.g. B. the so-called " Koch lysis ". The bacterial cell walls are destroyed in the course of a cell breakdown by adding lysozyme and the lysed bacteria are boiled for a short time or heated in a heating block at 95 ° C. The chromosomal DNA and the denatured proteins are centrifuged off. The plasmid DNA can be purified by means of phenol-chloroform extraction before precipitation. The DNA dissolved in water or TE buffer still contains RNA, which is why photometric quantification is not useful. If this is followed by a gel analysis , RNase A is added to the sample buffer.
Alternative procedures
Other general DNA purification methods are e.g. As the CsCl - density .
Individual evidence
- ↑ HC Birnboim, J. Doly: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. In: Nucleic Acids Res. (1979), Vol. 7 (6), pp. 1513-1523. PMID 388356 ; PMC 342324 (free full text).
- ↑ MA Marko, R. Chipperfield, HC Birnboim: A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. In: Anal Biochem. (1982), Vol. 121 (2), pp. 382-387. PMID 6179438 .
- ↑ R. Boom, CJ Sol, MM Salimans, CL Jansen, PM Wertheim-van Dillen, J. van der Noordaa: Rapid and simple method for purification of nucleic acids. In: J Clin Microbiol. (1990), Vol. 28 (3), pp. 495-503. PMID 1691208 ; PMC 269651 (free full text).
- ↑ TA Borodina, H. Lehrach , AV Soldatov: DNA purification on homemade silica spin columns. In: Anal Biochem. (2003), Volume 321 (1), pp. 135-137. PMID 12963065 .