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>> Protocols trong Sinh học Phân tử >>Plasmid và chọn dòng phân tử
Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali and SDS.
Plasmid DNA is isolated from intermediate-scale (20-50 ml) bacterial cultures by treatment with alkali and SDS.
Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with alkali and SDS.
Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains).
Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains).
Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks.
Large (>15 kb), closed circular plasmids are prepared (albeit inefficiently and in small yield) by lysing bacteria with SDS.
Crude preparations of plasmid DNA are first treated with lithium chloride and RNase (to remove RNA). The plasmid DNA is then precipitated in a solution containing polyethylene glycol and MgCl2.
The following table summarizes the salient features of many of the commercial resins that are currently available for plasmid purification. Individual manufacturers supply detailed instructions, which should be followed to the letter.
- Protocol: Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Continuous Gradients
Solutions containing plasmid DNA are adjusted to a density of 1.55 g/ml with solid CsCl. The intercalating dye, ethidium bromide, which binds differentially to closed circular and linear DNAs, is then added to a concentration of 200 µg/ml. During centrifugation to equilibrium, the closed circular DNA and linear DNAs form bands at different densities.
- [[Protocol: Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-
Ethidium Bromide Gradients: Discontinuous Gradients]] A solution containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44% w/v) is layered between two solutions of lesser (35% w/v CsCl) and greater density (59% w/v CsCl). During centrifugation to equilibrium, the closed circular plasmid DNA and linear DNAs form bands at different densities.
Ethidium bromide is removed from DNA by phase extraction with organic solvents.
Ethidium bromide is removed from DNA by chromatography through a cation-exchange resin.
- [[Protocol: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid
DNA by Centrifugation through NaCl]] Contamination of plasmid DNA by fragments of DNA and RNA is reduced to an acceptable level by centrifugation through 1 M sodium chloride. This method was devised by Brian Seed when he was a graduate student at Harvard University.
- [[Protocol: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid
DNA by Chromatography through Sephacryl S-1000]] Contamination of plasmid DNA by small fragments of nucleic acid is reduced dramatically by size-exclusion chromatography through Sephacryl S-1000.
- Protocol: Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Lithium Chloride
High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid DNA by high concentrations of LiCl and removed by low-speed centrifugation.
Thao tác kỹ thuật di truyền với các plasmid[sửa]
Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector.
Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms.
Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent E. coli. The maximum number of "correct" clones can generally be obtained from ligation reactions containing equimolar amounts of plasmid and target DNAs, with the total DNA concentration being <100 µg/ml. Blunt-end ligation catalyzed by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and polyamines such as spermidine.
During ligation in vitro, T4 DNA ligase will catalyze the formation of a phosphodiester bond between adjacent nucleotides only if one nucleotide carries a 5´-phosphate residue and the other carries a 3´-hydroxyl terminus. Recircularization of vector DNA can therefore be minimized by removing the 5´-phosphate residues from both termini of the linear, doublestranded plasmid DNA with alkaline phosphatase.
Linkers are small self-complementary pieces of synthetic DNA, usually 8-16 nucleotides in length, that anneal to form blunt-ended, double-stranded molecules containing a restriction site. Linkers are used to equip blunt-ended termini of DNA with restriction sites as an aid to cloning.
Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs.
Biến nạp plasmid[sửa]
- Protocol: The Hanahan Method for Preparation and Transformation of Competent E.coli: High-efficiency Transformation
This procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/mg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically.
- Protocol: The Inoue Method for Preparation and Transformation of Competent E.Coli: "Ultra-Competent" Cells
This protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/mg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C.
This protocol, developed approx. 30 years ago, is used to prepare batches of competent bacteria that yield 5 x 106 to 2 x 107 transformed colonies/µg of supercoiled plasmid DNA.
Electrocompetent bacteria are prepared by growing cultures to mid-log phase, washing the bacteria extensively at low temperature, and then resuspending them in a solution of low ionic strength containing glycerol. DNA is introduced during exposure of the bacteria to a short highvoltage electrical discharge.
Sàng lọc plasmid tái tổ hợp[sửa]
-complementation occurs when two inactive fragments of E. coli -galactosidase associate to form a functional enzyme. Many plasmid vectors carry a short segment of DNA containing the coding information for the first 146 amino acids of -galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of the enzyme. Although neither the host nor the plasmid-encoded fragments of -galactosidase are themselves active, they can associate to form an enzymatically active protein. Lac+ bacteria that result from - complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-gal. However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an amino-terminal fragment that is no longer capable of -complementation. Bacteria carrying recombinant plasmids therefore form white colonies. The development of this simple blue-white color test has greatly simplified the identification of recombinants constructed in plasmid vectors.
This procedure, a variant of the Grunstein and Hogness (1979) method, is used to screen a small number of bacterial colonies (<200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. After a period of growth, the colonies on the filter are lysed and processed for hybridization. The master plate is stored until the results of the screening procedure become available.
Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. This technique, a variant of the Grunstein and Hogness (1975) method, was developed at Cold Spring Harbor Laboratory in 1975. The procedure works best with 90-mm plates containing <2500 colonies.
This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial colonies (up to 2 x 104 colonies per 150-mm plate or 104 colonies per 90-mm plate).
In this protocol, based on the procedure of Grunstein and Hogness (1975), alkali is used to liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to the filter by UV-cross-linking or baking under vacuum.
This protocol describes procedures to hybridize DNA from transformed colonies immobilized on filters with radiolabeled probes and to recover from a master plate the corresponding colonies that hybridize specifically to the probe. The method is based on the procedure published by Grunstein and Hogness (1975).