miniprep

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2014/09/29 21:57:10」(JST)

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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:^{[citation needed]}

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

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 for example a gene encoding ampicillin or kanamycin resistance, which allows bacteria that have been successfully transformed to multiply uninhibited. 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. Bacteria are grown under favourable conditions.

It is important to use proper controls to make sure that the antibiotic is actually killing bacteria that have not taken up the plasmid. This can be accomplished by plating non-transformed cells on a plate with antibiotics. No growth is expected on that plate. Other systems of controls can also be helpful.

Harvesting and lysis of the bacteria

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 the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids 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.

Mini-preparation

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 50 to 100 µg depending on the cell strain. Miniprep of large number of plasmids can also be done conveniently by on filter paper lysing, the elution of the filter paper that contains plasmid can be directly sequenced to produce more than 700 bp high quality sequencing data with CE sequencing.^[1]

Midipreparation

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

Maxipreparation

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

Megapreparation

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.

Gigapreparation

The starting E. coli culture volume is 2.5-5 L of LB 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

^ High Throughput miniprep of plasmid for sequencing

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.

External links

http://www.protocol-online.org/prot/Molecular_Biology/Plasmid/Miniprep/
A miniprep procedure using diatomaceous earth to bind DNA during purification and washing.

English Journal

Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues.

Janecka A1, Adamczyk A2, Gasińska A2.
Analytical biochemistry.Anal Biochem.2015 May 1;476:8-10. doi: 10.1016/j.ab.2015.01.019. Epub 2015 Jan 30.
A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yiel
PMID 25640584

Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences.

Wagner Mackenzie B1, Waite DW1, Taylor MW1.
Frontiers in microbiology.Front Microbiol.2015 Feb 18;6:130. doi: 10.3389/fmicb.2015.00130. eCollection 2015.
The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variati
PMID 25741335

Rapid mitochondrial DNA isolation method for direct sequencing.

Quispe-Tintaya W1, White RR, Popov VN, Vijg J, Maslov AY.
Methods in molecular biology (Clifton, N.J.).Methods Mol Biol.2015;1264:89-95. doi: 10.1007/978-1-4939-2257-4_9.
Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequence results. Here, we describe a reliable method for the preparation of mtDNA-enri
PMID 25631006

Japanese Journal

Assessment of Small Scale Methods for Quick Extraction of Genomic DNA from Leaf Tissue of Sugarcane

SRIVASTAVA Sangeeta,GUPTA Prashant Shekhar
Cytologia : international journal of cytology 71(2), 181-188, 2006-06-25
Molecular studies to characterize genetic resources of sugarcane for use in breeding programmes require DNA extraction from several clones simultaneously so the DNA isolation procedures should be rapi …
NAID 10017390620

様々な野菜可食部組織を用いたDNAフィンガープリント法の開発(育種・遺伝資源)

荒木直幸,道後充恵,舟橋正隆 [他],山内直樹,執行正義
園芸学研究 4(2), 131-134, 2005-06-15
33種類の野菜可食部から, 微量サンプル抽出法で得られたタンパク質の混入度(A260/A280)は, いずれも1.8以上で, PCRに適する値が得られた.しかし, リーフレタス, ゴボウから得られたDNA溶液は褐色を呈し, PCR阻害がみられた.しかし, このような場合, 最終濃度10mMの2-メルカプトエタノールをDNA溶液に添加し, Nucleon PhytoPure Kitで精製することで, …
NAID 10016144986

様々な野菜可食部組織を用いたＤＮＡフィンガープリント法の開発

荒木直幸,道後充恵,舟橋正隆,山内直樹,執行正義
園芸学研究 4(2), 131-134, 2005
33種類の野菜可食部から, 微量サンプル抽出法で得られたタンパク質の混入度 (A260/A280) は, いずれも1.8以上で, PCRに適する値が得られた. しかし, リーフレタス, ゴボウから得られたDNA溶液は褐色を呈し, PCR阻害がみられた. しかし, このような場合, 最終濃度10mMの2-メルカプトエタノールをDNA溶液に添加し, Nucleon PhytoPure Kitで精製する …
NAID 130004476298

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[1] High Throughput miniprep of plasmid for sequencing

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