Plasmid preparation#Preparations by size

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have been transformed and isolated.{{Cite book | vauthors = Suza W, Lee D |url=https://iastate.pressbooks.pub/genagbiotech/chapter/recombinant-dna-technology/ |title=Genetics, Agriculture, and Biotechnology |publisher=Iowa State University |at= |language=en |chapter=11. Recombinant DNA Technology; Ligase enzyme and gene cloning|date=15 October 2021 }}{{cite journal | vauthors = Ismail R, Allaudin ZN, Lila MA | title = Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status | journal = Vaccine | volume = 30 | issue = 41 | pages = 5914–5920 | date = September 2012 | pmid = 22406276 | doi = 10.1016/j.vaccine.2012.02.061 | doi-access = free }} Virtually all plasmid vectors in common use encode one or more antibiotic resistance genes as a selectable marker, for example a gene encoding ampicillin or kanamycin resistance, which allows bacteria that have been successfully transformed to multiply uninhibited.{{Cite web |title=Plasmid |url=https://www.genome.gov/genetics-glossary/Plasmid |access-date=2022-12-10 |website=Genome.gov |language=en}}{{Cite book | vauthors = Batree L, Shriner W, Creech C |url=https://openoregon.pressbooks.pub/mhccmajorsbio/chapter/cloning/ |title=Principles of biology |publisher=Open Oregon Educational Resources |chapter=Biotechnology|year=2017 }}{{cite journal | vauthors = Bennett PM | title = Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria | journal = British Journal of Pharmacology | volume = 153 | issue = Suppl 1 | pages = S347–S357 | date = March 2008 | pmid = 18193080 | pmc = 2268074 | doi = 10.1038/sj.bjp.0707607 }} Bacteria that have not taken up the plasmid vector are assumed to lack the resistance gene, and thus only colonies representing successful transformations are expected to grow.{{cite journal | vauthors = Smalla K, Jechalke S, Top EM | title = Plasmid Detection, Characterization, and Ecology | journal = Microbiology Spectrum | volume = 3 | issue = 1 | pages = PLAS–0038–2014 | date = February 2015 | pmid = 26104560 | pmc = 4480600 | doi = 10.1128/microbiolspec.PLAS-0038-2014 }}

Bacteria are grown under favourable conditions.

There are several methods for cell lysis, including alkaline lysis, mechanical lysis, and enzymatic lysis.{{cite journal | vauthors = Rahman MM, Hosano N, Hosano H | title = Recovering Microalgal Bioresources: A Review of Cell Disruption Methods and Extraction Technologies | journal = Molecules | volume = 27 | issue = 9 | pages = 2786 | date = April 2022 | pmid = 35566139 | pmc = 9104913 | doi = 10.3390/molecules27092786 | doi-access = free }}{{cite journal | vauthors = Weber S, Grande PM, Blank LM, Klose H | title = Insights into cell wall disintegration of Chlorella vulgaris | journal = PLOS ONE | volume = 17 | issue = 1 | pages = e0262500 | date = 2022 | pmid = 35030225 | pmc = 8759652 | doi = 10.1371/journal.pone.0262500 | bibcode = 2022PLoSO..1762500W | doi-access = free }}{{cite journal | vauthors = Wang D, Li Y, Hu X, Su W, Zhong M | title = Combined enzymatic and mechanical cell disruption and lipid extraction of green alga Neochloris oleoabundans | journal = International Journal of Molecular Sciences | volume = 16 | issue = 4 | pages = 7707–7722 | date = April 2015 | pmid = 25853267 | pmc = 4425044 | doi = 10.3390/ijms16047707 | doi-access = free }}{{cite journal | vauthors = Borchers A, Pieler T | title = Programming pluripotent precursor cells derived from Xenopus embryos to generate specific tissues and organs | journal = Genes | volume = 1 | issue = 3 | pages = 413–426 | date = November 2010 | pmid = 24710095 | pmc = 6190294 | doi = 10.3390/mi8030083 | doi-access = free }}

The most common method is alkaline lysis, which involves the use of a high concentration of a basic solution, such as sodium hydroxide, to lyse the bacterial cells.{{cite journal | vauthors = Williams JA | title = Vector Design for Improved DNA Vaccine Efficacy, Safety and Production | journal = Vaccines | volume = 1 | issue = 3 | pages = 225–249 | date = June 2013 | pmid = 26344110 | pmc = 4494225 | doi = 10.3390/vaccines1030225 | doi-access = free }}{{Cite web | vauthors = Zazilek G |date=2010-04-12 |title=Alkaline Lysis |url=https://askabiologist.asu.edu/alkaline-lysis |access-date=2023-01-02 |website=askabiologist.asu.edu |language=en}}{{cite journal | vauthors = Birnboim HC, Doly J | title = A rapid alkaline extraction procedure for screening recombinant plasmid DNA | journal = Nucleic Acids Research | volume = 7 | issue = 6 | pages = 1513–1523 | date = November 1979 | pmid = 388356 | pmc = 342324 | doi = 10.1093/nar/7.6.1513 }} When bacteria are lysed under alkaline conditions (pH 12.0–12.5) both chromosomal DNA and protein are denatured; the plasmid DNA however, remains stable. 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 to lower the pH to around 7, the large and less supercoiled chromosomal DNA and proteins form large complexes and precipitate; but the small bacterial DNA plasmids stay in solution.

Mechanical lysis involves the use of physical force, such as grinding or sonication, to break down bacterial cells and release the plasmid DNA. There are several different mechanical lysis methods that can be used, including French press, bead-beating, and ultrasonication.

Enzymatic lysis, also called Lysozyme lysis, involves the use of enzymes to digest the cell wall and release the plasmid DNA. The most commonly used enzyme for this purpose is lysozyme, which breaks down the peptidoglycan in the cell wall of Gram-positive bacteria. Lysozyme is usually added to the bacterial culture, followed by heating and/or shaking the culture to release the plasmid DNA.

Plasmid preparation can be divided into five main categories based on the scale of the preparation: minipreparation, midipreparation, maxipreparation, megapreparation, and gigapreparation. The choice of which method to use will depend on the amount of plasmid DNA required, as well as the specific application for which it will be used.{{cite journal | vauthors = Serghini MA, Ritzenthaler C, Pinck L | title = A rapid and efficient 'miniprep' for isolation of plasmid DNA | journal = Nucleic Acids Research | volume = 17 | issue = 9 | pages = 3604 | date = May 1989 | pmid = 2726501 | pmc = 317816 | doi = 10.1093/nar/17.9.3604 }}{{cite journal | vauthors = Kovalenko SA, Tanaka M, Ozawa T | title = Simple methods for preparation of plasmid DNA yielding long and accurate sequence data | journal = Nucleic Acids Research | volume = 22 | issue = 25 | pages = 5771–5772 | date = December 1994 | pmid = 7838738 | pmc = 310149 | doi = 10.1093/nar/22.25.5771 }}

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 they are: 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 of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria.{{cite journal | vauthors = Chowdhury K | title = One step 'miniprep' method for the isolation of plasmid DNA | journal = Nucleic Acids Research | volume = 19 | issue = 10 | pages = 2792 | date = May 1991 | pmid = 2041760 | pmc = 328215 | doi = 10.1093/nar/19.10.2792 }}{{Cite web |title=Plasmid Mini-Prep {{!}} College of Biological Sciences |url=https://cbs.umn.edu/hackett-lab/protocols/mini-prep |access-date=2023-01-10 |website=cbs.umn.edu}} Commonly used miniprep methods include alkaline lysis and spin-column based kits.{{cite journal | vauthors = Zhang S, Cahalan MD | title = Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit | journal = Journal of Visualized Experiments | issue = 6 | pages = 247 | date = 2007-07-29 | pmid = 18997895 | pmc = 2557117 | doi = 10.3791/247 }} It is based on the alkaline lysis method. 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 5 to 50 μg depending on the cell strain.

Miniprep of a large number of plasmids can also be done conveniently on filter paper by lysing the cell and eluting the plasmid on to filter paper.

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

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

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

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

It is important to consider the downstream applications of the plasmid DNA when choosing a purification method. For example, if the plasmid is to be used for transfection or electroporation, a purification method that results in high purity and low endotoxin levels is desirable. Similarly, if the plasmid is to be used for sequencing or PCR, a purification method that results in high yield and minimal contaminants is desirable. However, multiple methods of nucleic acid purification exist.{{cite journal | vauthors = Tan SC, Yiap BC | title = DNA, RNA, and protein extraction: the past and the present | journal = Journal of Biomedicine & Biotechnology | volume = 2009 | pages = 574398 | date = 2009 | pmid = 20011662 | pmc = 2789530 | doi = 10.1155/2009/574398 | doi-access = free }}{{Cite web |title=Phenol-Chloroform Extraction {{!}} Herman Lab {{!}} Nebraska |url=https://hermanlab.unl.edu/protocols/phenolppt.html |access-date=2023-01-10 |website=hermanlab.unl.edu}} All work on the principle of generating conditions where either only the nucleic acid precipitates, or only other biomolecules precipitate, allowing the nucleic acid to be separated.

In high-throughput DNA extraction workflows, laboratory equipment such as [https://96wellplatetemplate.com/ 96 well plate template] can be utilized to efficiently process multiple samples in parallel. These templates allow for the automation of extraction protocols, significantly increasing the throughput of plasmid DNA isolation while maintaining consistency across large sample sets. When used in combination with automated liquid handling systems, a 96 well plate template helps streamline the process of extracting plasmid DNA from bacterial cultures, ensuring uniformity and reducing manual errors during the purification steps.

Ethanol precipitation is a widely used method for purifying and concentrating nucleic acids, including plasmid DNA.{{cite journal | vauthors = Zeugin JA, Hartley JL |year=1985 |title=Ethanol Precipitation of DNA |url=http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.56415.File.dat/Focus%20Volume%207%20Issue%204.PDF |journal=Focus |volume=7 |issue=4 |pages=1–2 |accessdate=2008-09-10}} The basic principle of this method is that nucleic acids are insoluble in ethanol or isopropanol but soluble in water. Therefore, it works by using ethanol as an antisolvent of DNA, causing it to precipitate out of solution and then it can be collected by centrifugation. The soluble fraction is discarded to remove other biomolecules.{{Cite web |title=Barrick Lab :: ProtocolsEthanolPrecipitation |url=https://barricklab.org/twiki/bin/view/Lab/ProtocolsEthanolPrecipitation |access-date=2023-01-10 |website=barricklab.org}}

Spin column-based nucleic acid purification is a method of purifying DNA, RNA or plasmid from a sample using a spin column filter.{{cite journal | vauthors = Ali N, Rampazzo RC, Costa AD, Krieger MA | title = Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics | journal = BioMed Research International | volume = 2017 | pages = 9306564 | date = 2017 | pmid = 28785592 | pmc = 5529626 | doi = 10.1155/2017/9306564 | doi-access = free }} The method is based on the principle of selectively binding nucleic acids to a solid matrix in the spin column, while other contaminants, such as proteins and salts, are washed away. The conditions are then changed to elute the purified nucleic acid off the column using a suitable elution buffer.

The basic principle of the phenol-chloroform extraction is that DNA and RNA are relatively insoluble in phenol and chloroform, while other cellular components are relatively soluble in these solvents. The addition of a phenol/chloroform mixture will dissolve protein and lipid contaminants, leaving the nucleic acids in the aqueous phase. It also denatures proteins, like DNase, which is especially important if the plasmids are to be used for enzyme digestion. Otherwise, smearing may occur in enzyme restricted form of plasmid DNA.

In beads-based extraction, addition of a mixture containing magnetic beads commonly made of iron ions binds to plasmid DNA, separating them from unwanted compounds by a magnetic rod or stand. The plasmid-bound beads are then released by removal of the magnetic field and extracted in an elution solution for down-stream experiments such as transformation or restriction digestion. This form of miniprep can also be automated, which increases the conveniency while reducing mechanical error.

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• {{cite journal | vauthors = Birnboim HC, Doly J | title = A rapid alkaline extraction procedure for screening recombinant plasmid DNA | journal = Nucleic Acids Research | volume = 7 | issue = 6 | pages = 1513–1523 | date = November 1979 | pmid = 388356 | pmc = 342324 | doi = 10.1093/nar/7.6.1513 }}

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• http://www.protocol-online.org/prot/Molecular_Biology/Plasmid/Miniprep/
• [http://www.thel4b.com/?page_id=1277 A miniprep procedure using diatomaceous earth to bind DNA during purification and washing.]

Category:Biological techniques and tools

Category:Genetics techniques

Category:Molecular biology