Subcloning of the target gene can be applied to (1) further analysis of the target gene and (2) gene recombination.
Operation method
Subcloning of target genes
Principle
The basic process of subcloning includes (1) preparation of target DNA fragments and vectors, (2) ligation of target DNA fragments and vectors, (3) transformation of ligation products, and (4) recombinant screening.
Materials and Instruments
E.coli JM109 Move I. Preparation of target DNA fragments and vectors Select the appropriate restriction endonuclease and digest the known target DNA and vector to obtain linear DNA for recombination. According to the specific conditions of the target DNA and vector, select one or two appropriate restriction enzymes to cut, respectively, to produce symmetric sticky ends (digested with one restriction endonuclease to produce with complementary protruding ends), asymmetric sticky ends (digested with two different restriction endonucleases to produce with non-complementary protruding ends), flat ends. In subcloning, asymmetric compatible end ligation is preferred, followed by symmetric sticky compatible end ligation, and flat end ligation is usually seldom used due to its low efficiency. However, sometimes the end of the target fragment does not match with the carrier, so the mismatched ends are usually flattened first, and then connected with flat ends. In vitro ligation of target DNA fragments and vectors using T4 DNA ligase. 1. Ligation requirements and results Exogenous DNA fragments with identical ends (flat or sticky) must be cloned into linear plasmid vectors with matching ends, but during the ligation reaction, both the exogenous DNA and the plasmid may undergo cyclization, and tandem oligomers may also be formed. Therefore, the concentrations of the two DNAs in the ligation reaction must be carefully adjusted to optimize the amount of the "correct" ligation product, and alkaline phosphatase is often used to remove the 5' phosphate group to inhibit auto-cyclization of the vector DNA. T4 DNA ligase is used to perform the in vitro ligation of the target DNA fragment to the vector, i.e., to form a new covalent bond between the 5' phosphate of the double-stranded DNA and the adjacent 3' hydroxyl group. If both strands of the vector with 5'phosphate (not dephosphorylated), can form four new phosphodiester bonds; such as vector DNA has been dephosphorylated, can only form two new phosphodiester bonds, this time the resulting recombinant DNA with two single-stranded gaps, can be repaired after importing into the receptor cell. 2.T4 DNA ligase on the purpose of DNA fragments and vector ligation of the general program 1. The ligation reaction is generally carried out in a sterilized 0.5 ml centrifuge tube. 2. In a 10μl volume of the reaction system: take 50-100ng of vector, add a certain proportion of exogenous DNA molecules (generally the molar number of linear vector DNA molecules and exogenous DNA molecules is 1:1-1:5), and make up ddH2O to 8μl. 3. Mix gently, centrifuge for 5 min at 56℃, and then quickly transfer to ice bath. 4. Add 1 μl of 10×Buffer containing ATP, appropriate unit of T4 DNA ligase, make up to 10 μl with ddH2O, slightly centrifuged, and ligated at the appropriate temperature (generally 14-16℃ water bath) for 8-14hr. Transformation of ligation products 1. Preparation of receptor cells (1) Preserve DH5α (or other strains) at -70℃, inoculate the bacteria with an inoculating loop on a 1.5% agar plate, and invert the culture at a constant temperature of 37℃ until a single colony appears (about 14-16 hr). (2) Pick single colonies, inoculate them in 2.0 ml LB liquid medium, incubate at constant temperature of 37℃ with 250g shaking overnight (about 12hr). (3) Take 0.5ml of overnight culture solution, inoculate in 100ml LB liquid medium, 37℃ shaking culture 2-2.5hr, to OD600 is 0.4-0.5, placed in 4℃ refrigerator cooling 1-2hr. (Note: All the following operations should be carried out in an ice bath.) (4) Divide the culture solution into two 50ml centrifuge tubes, centrifuge at 4℃, 4000g×10min, discard the supernatant, and suspend with 25ml of 0.1M MgCl2 in an ice bath for 30min. (5) Centrifuge at 4℃,4000g×10min,discard the supernatant and add 1ml of 0.1M CaCl2-glycerol solution in ice bath for suspension. (6) Dispense 100μl/tube into 1.5ml centrifuge tube and freeze at -70℃ for spare. Note: This method of preparation of sensory cells, can make each microgram of super-helical plasmid DNA to produce 5 × 106-2 × 107 colonies, such transformation efficiency is sufficient to meet the needs of all the conventional cloning in the plasmid, the preparation of sensory cells can be stored at -70 ℃, but the preservation of too long a period of time will make the transformation efficiency to a certain extent affected, generally three months or less the transformation efficiency does not change much. 2. Transformation of connection products (1) Take 100 μl stored in -70 ℃ calcineurin, ice bath melt away; (2) Add the appropriate amount of connection product (generally no more than 10μl, gently mixed, ice bath for 20min; (3) heat shock at 42 ℃. (3) Heat shock at 42 ℃ for 90s, quickly transferred to the ice bath, continue ice bath for 2-3min. (4) Add LB liquid medium 200μl, incubate at 37℃ with slow shaking for 45min. (5) Apply appropriate amount of culture to 1.5% agar LB plate (add antibiotics or/and X-Gal/IPTG according to the nature of the plasmid), wait until there is no liquid flow on the surface of the gel, incubate in an inverted incubator at 37℃ for 12-16hr. Screening of recombinants Screen recombinants according to the genetic characteristics of the vector, such as α-complementary, antibiotic genes and so on. Many vectors used nowadays carry a short segment of E. coli DNA with the regulatory sequence of the β-galactosidase gene (lacZ) and coding information for the first 146 amino acids. A multiple cloning site (MCS) is inserted into this coding region, which does not disrupt the reading frame but allows a few amino acids to be inserted into the amino terminus of β-galactosidase without affecting function, and this vector is suitable for use in host cells that can encode a portion of the C-terminal sequence of β-galactosidase. Thus, although neither the host nor the plasmid-encoded fragments are enzymatically active, they can form enzymatically active proteins when present together. In this way, the lacZ gene achieves complementation, called α-complementation, between a host cell lacking the proximity-manipulated gene region and a plasmid with an intact proximity-manipulated gene region. LacZ+ bacteria resulting from α-complementation are easily recognized by producing blue colonies in the presence of the chromogenic substrate X-Gal in the presence of the inducer IPTG. However, when exogenous DNA is inserted into the polyclonal site of the plasmid, it almost inevitably results in an amino-terminal fragment that is not α-complementary, allowing bacteria with the recombinant plasmid to form white colonies. This screening of recombinants is also known as blue-white spot screening. If blue-white spot screening is used then bacteria with recombinant plasmids form white colonies after inverted incubation of Calcitonella plates transformed with ligation products for 12-16hr in a 37°C temperature chamber. Caveat 1. When preparing, recovering and purifying the target DNA fragments, foreign DNA contamination should be avoided. 2. The reaction conditions of T4 DNA ligase produced by different manufacturers are slightly different, but there are optimal reaction conditions in their product manuals, including the amount of T4 DNA ligase, the temperature and time of action for the connection of DNA molecules with different terminal properties. At the same time, ligase buffer (10×, 5×, 2×) is provided, which contains ATP at the required concentration, and its decomposition should be avoided by placing it at high temperature and repeated freezing and thawing. 3. Transformation of ligated products: Bacterial cells have the ability to receive exogenous DNA under appropriate conditions after treatment with special reagents, therefore, the above ligated products can be transformed into receptor cells by thermal stimulation or electric pulse, and the imported recombinant DNA will be proliferated when the bacteria proliferate in large quantities at the same time. 4. Centrifuge tubes and culture flasks used for the preparation of sensory cells should preferably be treated with acid and alkali or new ones should be used, and autoclaved at 15 lbf/in2 for 20 min. 5. Whether the inserted fragments of the recombinant plasmid in the white colonies are the target fragments should be identified. For more product details, please visit Aladdin Scientific website.
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