Electroshock transformation experiments in Escherichia coli
Electroshock transformation experiments in Escherichia coli
Electroshock transformation experiments in E. coli can be used for: easier and faster transformation.
Operation method
Electroshock transformation experiments in Escherichia coli
Principle
Transformation is a phenomenon in which a cell of one genotype absorbs DNA from a cell of another genotype from the surrounding medium and changes its genotype and phenotype accordingly. The phenomenon was first discovered in bacteria. It is also one of the earliest discovered forms of genetic material transfer between bacteria, and is different from transduction, in which genetic material is passed on through phage infection, and bacterial splicing, in which DNA is transferred through contact between bacterial cells.
Materials and Instruments
Plasmid DNA Move I. Material preparation Caveat The same bacteria can also change transformation efficiency due to a change in genotype. Bacterial restriction endonucleases are able to break down foreign DNA, so transformation efficiency can be improved if mutant strains with inactivated restriction enzymes are used as transformation recipients. Mutants with significantly reduced transformation efficiency can also be obtained by screening, including mutants with reduced adsorption, uptake and integration capacities. Some of the mutants with reduced transformation efficiency are particularly sensitive to ultraviolet light, a property that is also characteristic of many mutants that have lost the ability to repair DNA damage and recombination, which shows that the integration of DNA and repair of DNA damage and recombination in the transformation process involves some of the same enzymes. Common Problems Bacteria with identical genotypes can change their transformation efficiency due to a change in physiological state. The physiological state of being able to take up exogenous DNA is called the sensory state. In many bacteria, the sensory state appears rapidly during the late logarithmic growth period and disappears after a period of time. Bacteria in the sensory state can be transformed 10,000 times more efficiently compared to bacteria in the non-sensory state. For more product details, please visit Aladdin Scientific website.
Glycerol Pure water SOC Culture medium
CYT LB SOB Agar Plate Electroshock Instrument Electroshock Cup Ice Bath Box Liquid Nitrogen
1. Buffers and solutions
Glycerol (10% V/V) (molecular biology grade), pre-cooled on ice.
Pure water (Milli-Q grade or equivalent, filtered with pre-treated Nalgene filter membrane (0.45 μm pore size) to remove bacteria, place on ice.
2. Nucleic acids and oligonucleotides
Plasmid DNA ( recombinant plasmid)
3. Media
(1) CYT, place on ice.
10% ( V/V) glycerol, 0.125% ( m/V) yeast powder, 0.25% ( m/V) peptone, this recipe is from Tung and Chow (1995).
(2) LB, pre-warmed to 37°C
(3) SOB agar plates containing 20 mmol/L MgSO4 and appropriate antibiotics.
Standard SOB agar plates contain 10 mmol/L MgSO4.
(4) SOC culture solution
Approximately 1 ml is required for each transformation reaction.
(5) Specialized equipment
Shock meter and shock cups with electrodes spaced at 0.1-0.2 cm.
Ice bath box
Liquid nitrogen
II. METHODS
1. Cell preparation
(1) Pick a single colony of E. coli from a fresh agar plate, inoculate it into a conical flask containing 50 ml of LB culture medium, and incubate it at 37 ℃ with shaking (250 r/min on a shaker) overnight.
(2) Inoculate two 25 ml overnight cultures into 2 L conical flasks containing 500 ml of pre-warmed LB medium, incubate at 37℃ with shaking (300 r/min), and measure the OD600 value every 20 min.
(3) When the OD600 value reaches 0.4, quickly place the culture in an ice bath for 15~30 min, shaking slowly to ensure that the contents are sufficiently cooled. Pre-cool the centrifuge tube on ice to prepare for the next step.
(4) Transfer the bacteria to an ice-cold centrifuge tube and centrifuge at 1000 g ( 2500 r/min ) for 15 min at 4°C with a SorvalI GSA turntable (or equivalent) to recover the cells. Pour off the culture medium and resuspend the precipitate with 500 ml of ice-cold purified water.
(5) Centrifuge the cells at 1000 g (2500 r/min) for 20 min at 4°C with a Sorvall GSA turntable (or equivalent) to recover the cells. Pour off the culture medium and resuspend the precipitate with 250 ml of ice-cold 10% glycerol.
(6) Recover the cells by centrifugation at 1000 g (2500 r/min) for 20 min at 4°C with a Sorvall GSA turntable (or equivalent). Pour off the culture medium and resuspend the precipitate with 10 ml of ice-cold 10% glycerol.
(7) Recover the cells by centrifugation at 1000 g (2500 r/min) for 20 min at 4°C with a Sorvall GSA turntable (or equivalent). Carefully pour off the supernatant, and then suck off any residual liquid from the walls of the tube with a Pasteur pipette attached to a vacuum. Resuspend the precipitate by adding 1 ml of ice-cold GYT culture medium.
(8) Measure the OD600 value after 100-fold dilution of the above suspension. Dilute with ice-cold GYT culture medium to a concentration of 2X1010~3X1010 cells/ml (1. 0OD600= ~2. 5X1010 cells/ml).
(9) Transfer 40 μl of the above suspension to an ice-cold electroshock cup (~0.2 cm gap) of the electrotransfer apparatus, and check whether there is a short circuit during the shock. If there is, then the remaining cell suspension is washed once with ice-cold GYT culture solution so that the conductance of the bacterial suspension is low enough (<5 mEq ).
(10) If the freshly prepared electrokinetic sensory cells are to be used immediately, proceed to step (12). For freezing at -70°C, dispense the suspension at 40 μl per portion into cooled sterile 0.5 ml microcentrifuge tubes, seal the tubes tightly, and rapidly freeze the sensory cells in liquid nitrogen. Store at -70℃ for backup.
(11) When frozen electroconverted sensory cells are required, remove the appropriate number of portions from the -70°C freezer, allow to thaw at room temperature, and then transfer the tubes to ice.
2. Electroshock Conversion
(12) Aspirate 40 μl of freshly prepared (or frozen-thawed) sensory cells into 0.5 ml ice-cold sterile microcentrifuge tubes. Cool on ice together with the electroconverter cuvette.
(13) Add 10 pg to 25 ng of DNA to be transformed to each microcentrifuge tube in a volume of 1 to 2 μl and place on ice for 30 to 60 s. Include all positive and negative controls.
DNA transformed by electroshock should preferably be dissolved in water or TE (pH 8.0) in the concentration range. If the DNA ligand is to be directly shock transformed, both options are possible. Either the ligand can be diluted 10-20 times in H2O, or the DNA can be purified by centrifugal chromatography or extracted by phenol-chloroform extraction and ethanol precipitation. The precipitated DNA should be washed with 70% ethanol and dissolved in either TE (pH 8.0) or H2O at a concentration of 1-10 μg/ml.
A high efficiency of transformation is necessary for the construction of the library, while co-transformation is necessary for the construction of the library. The total concentration of DNA should be less than 10 ng/ml (Dower et al., 1988), and for transformation of E.coli with a superhelical plasmid, a conventional transformation of 10-50 pg of DNA is sufficient. If transformation is performed with a subcloned plasmid, the ligation product should be diluted to a concentration of 25 ng of DNA.
(14) Adjust the electroshock meter so that the electrical pulse is 25 μF, voltage 2.5 kV, and resistance 200 Ω.
(15) Add the bacteria and DNA mixture to the cold electroshock cup, tapping the liquid to ensure that the bacteria and DNA suspension are at the bottom of the electroshock cup. Wipe off any condensation and mist from the outside of the electroshock tank. Place the stun cup into the stunner.
(16) Initiate the electrical impulse to the cells according to the parameters set above. The instrument should display an electric field strength of 12.5 kV/cm for 4 to 5 ms.
The ions in the stun cup increase the conductivity of the solution and generate an electric current and an arc or discharge in the solution containing the cells and DNA. When an electrical pulse is emitted, the presence of a popping noise in the shock tank makes the solitary light usually appear noticeable. Uneven charge transfer in the shock tank can greatly reduce conversion efficiency. Arcing occurs when the conductance of the solution is greater than 5 mEq (10 mmol/L salt or 20 mmol/L Mg2+ solution), and can also be induced by high temperatures. If the arcs are generated only in the presence of DNA, the ions should be removed during DNA preparation at step 13.
(17) At the end of the pulse, remove the cuvette as quickly as possible and add 1 ml of SOC culture solution at room temperature.
Some researchers believe that the addition of culture solution at room temperature can cause thermal excitation and thus increase transformation efficiency.
(18) Transfer the cells to 17X100 mm or 17X150 mm polyethylene tubes and incubate at 37°C for 1 h under gentle shaking.
(19) Take different volumes (up to 200 μl per 90 mm plate) of electroshock-transformed cells and spread them on SOB agar plates containing 20 mmol/L MgSO4 and appropriate antimicrobials.
With superhelical plasmid transformation, it can be expected that there will be many transformants, so a sterile inoculation loop is used to inoculate a small amount of bacterial fluid to line the agar plate (or a localized portion of the plate) containing the appropriate antimicrobials. However, if a small number of transformants is expected, it is best to inoculate 5 plates with 200 μl of bacterial solution per plate. Since the large number of dead cells produced during electroshocking has an inhibitory effect on the growth of the sparse transformants, we do not recommend the method of inoculating only one plate with concentrated bacterial solution.
(20) Allow the liquid in the culture plate to be completely absorbed at room temperature.
(21) Invert the culture plate and incubate at 37℃, the transformed clones should appear within 12~16 h. The plate should be incubated at 37℃, and the transformed clones should appear within 12~16 h.
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.