Ligation, transformation and screening of recombinant plasmids
Ligation, transformation and screening of recombinant plasmids
Recombinant plasmid ligation, transformation and screening are mainly used to obtain recombinants containing the target gene linkage.
Operation method
Ligation, transformation and screening of recombinant plasmids
Principle
The vector DNA and exogenous DNA fragments were cut with specific restriction endonucleases and purified, and the two were ligated in vitro (if T-vectors were used, they could be ligated directly with the purified PCR products), and the recombinants were selected by using the principle of blue-white-spotting screening after transformation of the host bacteria. Since the vector carries Amp' and lacZ genes, the recombinants are screened by a combination of Amp resistance screening and α-complementation principle screening. Preliminary resistance screening: the vector carries the Amp' gene but not the exogenous fragment, so after transformation of the recipient bacteria, only the transformants with the vector DNA can survive on the LB plate containing Amp, while the transformants with the exogenous fragment cyclized by itself cannot survive. Further blue and white spot screening: the vector carries the regulatory sequence of the β-galactosidase gene (lacZ) and the coding sequence for the 146 amino acids at the N-terminus of β-galactosidase. A polyclonal site was inserted in this coding region, but did not disrupt the reading frame of lacZ and did not affect its normal function. e.coli DH5α, Top10, or JM series strains carried the coding information for the C-terminal part of the sequence of β-galactosidase. In their respective independence, neither the vector nor the strains encoded fragments of β-galactosidase were enzymatically active. However, when the vector and the strain are fused together, a protein with enzymatic activity can be formed. This phenomenon of complementation between a mutant with a deletion of the near-manipulated gene region on the lacZ gene and a β-galactosidase mutant with an intact near-manipulated gene region is called α-complementation. Lac+ bacteria produced by α-complementation are easier to identify, and it is induced to form blue colonies by IPTG (isopropylthio-β-D-galactoside) in the presence of the color-generating substrate X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). When the exogenous fragment is inserted into the polyclonal site of the vector it leads to a change in the reading frame, inactivation of the expressed protein, and loss of α-complementary ability of the resulting amino acid fragments, so that the transformants containing the recombinant plasmid under the same conditions can only form white colonies on the color-biased induction medium.
Materials and Instruments
Exogenous DNA Fragments Move I. Ligation reaction Transfer about 10 ng of vector DNA into a sterile microcentrifuge tube, add at least 4 times the molar amount of exogenous DNA fragments, then add 1 μL of 10 × T4 DNA ligase buffer (ligase buffer), 0.5 μL - 1 μL of T4 DNA ligase, and add sterile ultrapure water to a volume of 10 μL. Add sterile ultrapure water to a volume of 10 μL, mix well and then collect all the liquid to the bottom of the tube by microcentrifuge, and keep warm at 16℃ overnight. Transformation of E.coli DH5α receptor cells 1. Take 200 μL (50 μL for high efficiency) of receptor cell suspension from a 70℃ refrigerator and thaw on ice. 2. 2. After thawing, add 1IxL-51xL of ligation product solution, flick and mix well, and leave on ice for 30rain. 3. 3. Incubate in a 42℃ water bath for 90s and quickly cool on ice for 3min--5min. 4. 4. Add lmL of LB liquid medium (without Amp) to the tube, mix well and incubate at 37°C with shaking for lh to allow the bacteria to resume growth and express the plasmid-encoded antibiotic resistance gene (Amp'). 5. Shake the above bacterial solution well and take 100 μL (if the receptor efficiency is low or the connection efficiency is low then lmL of bacterial solution can be collected in 4500 rpm low speed centrifuge all the bacterial bodies and resuspended in 100 μL of LB culture medium) coated in Amp-containing screening plate, placed face up for 30min, until the bacterial solution is completely absorbed by the medium, inverted the dish and placed it in 37 ℃ to incubate for 16h --① Do three controls at the same time. ①Make three controls at the same time Control 1: Replace the DNA solution with an equal volume of sterile double-distilled water, take only 5μL of bacterial solution and spread it on the LB plate without antibiotics, and this group should produce a large number of colonies under normal circumstances. Control 2: Replace the DNA solution with an equal volume of sterile double-distilled water, and take only 5μL of the bacterial solution to spread on the LB plate containing antibiotics when coating the plate. No colonies should normally appear in this group. Control 3: Take a certain amount of known concentration of control plasmid DNA (for the ligation vector plasmid in this experiment) and add it to an equal number of receptor cells, and other operations are the same as those for the ligation system. This control should show a larger number of colonies on the antibiotic-containing LB plate under normal conditions. ② Count the number of colonies in each petri dish The colonies that grow on the antibiotic-containing plate after transformation are the transformants, and the total number of transformants and transformation frequency can be calculated according to the number of colonies in the dish with the following formula: Total number of transformants=number of colonies×dilution×total volume of transformation reaction stock solution/volume of coated plate stock solution Transformation frequency (number of transformants/mg plasmid DNA) = total number of transformants/plasmid DNA added (mg) Total number of sensory cells = number of colonies in control group 1 x number of dilutions x total volume of stock solution / volume of coated plate stock solution Receptor cell transformation efficiency = total number of transformants/total number of receptor cells Screening and identification of recombinant plasmids The transformants with empty vector of T-vector are blue colonies on X-gal and IPTG medium due to β-galactosidase activity; the transformants with recombinant plasmid lose β-galactosidase activity due to the insertion of exogenous DNA fragments, and thus are white colonies on X-gal and IPTG medium. 1. Plasmid electrophoresis for preliminary identification of recombinants White single colonies were picked with a sterile toothpick and inoculated into 5mL LB liquid medium containing Amp 50μg/mL, and incubated with shaking at 37℃ for 12 h. Plasmid DNA was prepared in small quantities by alkaline lysis for electrophoresis, and a T-vector was prepared at the same time as a control, and the mobility of recombinant plasmid with inserted fragments was slower than that of the T-vector during electrophoresis. 2. Plasmid digestion to determine recombinants Use the restriction endonuclease corresponding to the unterminated end of the linkage to further digest the plasmid with slower migration rate in the above electrophoresis, and at the same time, perform control digestion on the empty vector, if the plasmid is indeed inserted with the exogenous DNA fragments, you can see the DNA bands in the electrophoresis which are the same size as the inserted fragments. Caveat 1. T4 DNA ligase (T4 DNA ligase) and ligation buffer (10×), purchased from the company. 2. corresponding endonuclease (for enzymatic fragmentation and vector and subsequent identification of recombinants), purchased from the company. 3. X-gal reservoir solution (20mg/mL): buy the solution directly, or dissolve the X-gal solid in dimethylformamide to make a 20mg/mL solution.20mg/mL reservoir solution, wrapped with aluminum foil or black paper to prevent destruction by light, and stored at -20℃. 4. IPTG storage solution (200mg/mL): dissolve 200mg of IPTG in 800μL of distilled water, and then dilute to 1mL with distilled water.After dissolving 200 mg of IPTG in 800 μL of distilled water, the solution was diluted with distilled water to 1 mL, filtered with 0.22 μm filter membrane to remove bacteria, dispensed into centrifuge tubes and stored at -20℃. 5. Screening medium containing X-gal and IPTG: Before coating the transformants, add 100 μg/mL of Amp to the surface of a prepared LB plate.Before coating the transformants, add 40mL of X-gal reservoir solution and 4μL of IPTG reservoir solution to the surface of a pre-prepared LB plate containing 100μg/mL of Amp and spread the solution with a sterile glass applicator, and place it at 37℃ for 3h and 4h so that the liquid on the surface of the medium is completely absorbed. 6. Alkaline lysis method to prepare plasmid reagents in small quantities. 7. specific restriction endonucleases and electrophoresis reagents. Common Problems Common Problems Analysis and Treatment: Since this experimental process involves several experimental aspects, such as the efficiency of various aspects of enzyme digestion, ligation, receptor cells, transformation, etc., some problems often occur in the experiment. Such as: 1. There are a large number of colonies on control 1; no colonies on control 2; no colonies or a small number of colonies on control 3; no colonies on the transformation plate of the connection system. There are no colonies on the plate. This may be due to the low efficiency of the sensory cells, so reprepare a high-efficiency sensory state. 2. large number of colonies on control 1; no colonies on control 2; large number of colonies on control 3; no colonies on ligated transformation plate. No colonies. This may indicate that the ligation efficiency is too low. Re-adjust the ligation system to improve the ligation efficiency. 3. Control 1 has a large number of colonies; control 2 also has colonies; control 3 has colonies; the ligation system has colonies on the transformation plate. There are colonies on control 1; there are colonies on control 2; there are colonies on control 3; and there are colonies on the transformation plate of the ligation system. This may indicate that the susceptor cells are contaminated and that a new original susceptor cell line was used to prepare the susceptor. 4. control 1 has a large number of colonies; control 2 has no colonies; control 3 has a relatively large number of colonies; the linkage system transformation plate has a moderate number of colonies, but only a few colonies. There are a moderate number of colonies, but only blue colonies, no white colonies. It may indicate that the efficiency of the ligation system is too low, and the transformed plasmids are vector self-linking plasmids (T vector; or the enzyme digestion of the vector is incomplete, and the isolated and purified vector contains fragments that have not been double enzyme digested), so re-prepare the fragments and vectors, and adjust the ligation system to improve the efficiency of the ligation. 5. source "Experimental techniques in molecular biology". For more product details, please visit Aladdin Scientific website.
Ligation Buffer T4 DNA Ligase X-gal Reservoir IPTG Reservoir McConkey's Selective Medium
Constant temperature shaker Benchtop high speed centrifuge Constant temperature water bath Electrophoresis device Electrothermal incubator Electrophoresis instrument Pipette gun eppendorf tube