Inoue's method (1990) for the preparation of E. coli receptor cells is good enough to achieve even the transformation efficiency of Hanahan's method (1980). However, transformation efficiencies of 1X108 to 3X108 transformed clones/μg of plasmid DNA are more commonly achieved under standard laboratory conditions. The advantage of this method over the Hanahan method is that it is not overly detailed, but is more reproducible and predictable. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Inoue method experiments for the preparation and transformation of susceptible Escherichia coli (preparation of super-susceptible cells)
Principle
Inoue's method (1990) for the preparation of E. coli receptor cells is good enough to reach the transformation efficiency of Hanahan's method (1980). However, transformation efficiencies of 1X108 to 3X108 transformed clones/μg of plasmid DNA are more commonly achieved under standard laboratory conditions. The advantage of this method over the Hanahan method is that it is not overly detailed, but is more reproducible and predictable.
Materials and Instruments
Plasmid DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Dimethyl sulfoxide DMSO Inoue Transformation Buffer
SOB Agar plate SOC medium Sorvall GSA Turn head or equivalent Liquid nitrogen Polypropylene centrifuge tube Water bath
1. Buffers and solutions
(1) Dimethyl sulfoxide DMSO
The oxidation product of dimethyl sulfoxide is presumed to be dimethyl sulfide, an inhibitor of conversion (Hanahan 1985). To avoid these problems, high quality DMSO should be purchased.
(2) Inoue transformation buffer
Pre-cool to 0°C on ice before use.
2. Nucleic acids and oligonucleotides
Plasmid DNA (recombinant plasmid)
3. culture media
(1) LB or SOB medium (for initial bacterial growth)
(2) SOB agar plates containing 20 mmol/L MgSO4 and appropriate antibiotics.
Standard SOB agar plates contain 10 mmol/L MgSO4.
(3) SOB medium (to culture transformed bacteria)
Before incubation, prepare three 1 L conical flasks containing 250 ml of 18-20 ℃ SOB.
(4) SOC culture medium
About 1 ml is required for each transformation reaction.
4. Centrifuge and rotary head
Sorvall GSA turntable or its equivalent
5. Specialized equipment
Liquid nitrogen, polypropylene tubing, 18°C shaker, water bath with adjustable temperature of 42°C.
Methods
1. Preparation of sensory cells
(1) Prepare Inoue Transformation Buffer (pre-cool on ice before use).
Prepare 0.5 mol/L PIPES (pH 6.7) [piperazine-N, N'-bis(2-ethanesulfonic acid)] solution: dissolve 15.1 g of PIPES in 80 ml of water (Milli-Q grade, or equivalent), adjust the pH to 6.7 with 5 mol/L KOH, and then add purified water to 100 ml. The solution was filtered with pre-treated Nalgene filter membrane (0.45 μm pore size) to remove bacteria. The sample was divided into small portions and stored at -20℃.
② Preparation of Inoue transformation buffer: Dissolve the following components in 800 ml of purified water, then add 20 ml of 0.5 mol/L PIPES (pH 6.7), then add 20 ml of 0.5 mol/L PIPES (pH 6.7), and then add purified water to make it to 1L. 
(iii) Filter with pre-treated Nalgene filter membrane (0.45 μm pore size) to remove bacteria. The sample was divided into small portions and stored at -20℃.
(2) Pick a single colony (2~3 mm in diameter) from a petri dish incubated at 37℃ for 16~20 h, inoculate it into 25 ml LB culture medium or SOB culture medium in a 250 ml conical flask, and incubate it at 37℃ for 6~8 h on a shaker (250~300 r/min).
(3) At about 6 o'clock in the evening, inoculate the above initial cultures into three 1L conical flasks containing 250 ml of SOB culture medium, the first with 10 ml, the second with 4 ml, and the third with 2 ml, and incubate at 18~22℃ in a medium-speed shaker overnight.
(4) In the morning of the next day, the OD600 values of the three cultures were measured every 45 min.
(5) When the OD600 of one bottle was 0.55, put the bottle on ice for 10 min, and discard the other two bottles.
Due to the large temperature difference between day and night in most laboratories, and the temperature difference also varies with the seasons of the year and the number of staff. Therefore, it is difficult to predict the period of appropriate OD values, especially since simultaneous incubation of three bottles with different concentration gradients at night improves the chances of success, with at least one bottle incubated overnight being compliant.
(6) Collect the organisms by centrifugation at 4°C for 10 min at 2500 g (equivalent to Sorall GSA turning head, 900 r/min).
(7) Pour off the culture solution, invert the centrifuge tube onto blotting paper for 2 min to absorb the remaining liquid, and use a vacuum aspirator to suck up the medium adhering to the wall of the tube.
(8) Resuspend the bacterial precipitate with 80 ml of pre-cooled Inoue's conversion buffer.
Swirl gently, do not mix with an oscillator or pipetting.
(9) Centrifuge at 2500 g at 4°C (equivalent to a Sorall GSA turntable at 3900 r/min) for 10 min to collect the organisms.
(10) Pour off the top layer of culture medium, invert the centrifuge tube onto blotting paper for 2 min to absorb the remaining liquid, and use a vacuum aspirator to suck up the culture medium attached to the wall of the tube.
2. Freezing of sensory cells
(1) Gently resuspend the precipitate with 20 ml of pre-cooled Inoue Transformation Buffer.
(2) Add 1.5 ml of DMSO. gently mix the bacterial suspension and place on ice for 10 min.
(3) Quickly dispense the suspension into cooled sterile microcentrifuge tubes, seal the tubes tightly, and freeze the sensory cells in liquid nitrogen. Store at -70℃ for preparation.
(4) When needed, remove a tube of sensory cells from the refrigerator, hold the tube in the palm of your hand and melt the cells. As soon as the cells are melted, transfer the tube to an ice bath and leave it on ice for 10 min.
(5) Using a cold sterile pipette tip, transfer the sensory cells to a cooled sterile polypropylene tube (17x170 mm) and place on an ice bath.
Glass tubes are not used because they reduce the transformation efficiency by about 10-fold.
3. Transformation
Includes positive and negative controls.
(1) Add the DNA fragments to be transformed to the tube containing the sensory cells (25 ng of DNA for 50 μl of sensory cells), the volume should not exceed 5% of the sensory cells, and gently rotate the tube several times to mix the contents. There should be at least one control tube in the experiment: one containing the sensory cells and a known amount of superhelical plasmid DNA, and the other containing only sensory cells. Mix the contents well and ice bath for 30 min.
(2) Place the tubes in a circulating water bath preheated to 42°C and leave for exactly 90 s without shaking.
Heat excitation is a critical step and it is important to reach the heat excitation temperature accurately. The temperatures and incubation times shown here are for measurements using Falcon 2059 tubes; other types of tubes may give different results.
(3) Quickly transfer the tubes to an ice bath and allow the cells to cool for 1~2 min.
(4) Add 800 μl of SOC medium to each tube, warm the medium to 37°C using a water bath, then transfer the tubes to a shaker set at 37°C and incubate for 45 min to allow the bacteria to recover and express the plasmid-encoded antibiotic resistance marker gene.
To maximize the transformation rate, the cells should be shaken gently (< 225 r/min) during the recovery period.
If with α-complementary, please see "Screening Bacterial Colonies with X-gal and IPTG: α-complementary".
(17) Transfer the appropriate volume (up to 200 μl per 90 mm plate) of transformed sensory cells to SOB medium containing 20 mmol/L MgSO4 and the appropriate antibiotic.
If tetracycline is used as a selection marker, the entire transformation mixture can be spread on a separate dish (or in soft agar), and can be centrifuged in a microcentrifuge at room temperature for 20 s to collect the transformed organisms, and 100 μl of SOC can be added to resuspend the precipitate while tapping the walls of the tube.
IMPORTANT: The glass spreader needs to be soaked in ethanol and then cauterized on an alcohol lamp and allowed to cool to room temperature before the transforming bacteria can be gently spread on the surface of the agar plate.
If testing for ampicillin resistance, the transformants should be spread at a low density (no more than 104 colonies per 90 mm plate) and incubated at 37°C for no more than 20 h. Cyanobenzylpenicillin-resistant transformants may secrete β-lactamase into the medium, which rapidly inactivates penicillin in the area surrounding the colony. In this way, too high a density when spreading plates or too long an incubation time can lead to the emergence of satellite colonies that are sensitive to cyanobenzylpenicillin soxhlet. The use of carboxybenzylpenicillin instead of ampicillin in the selective medium, as well as an increase in the antibiotic concentration from 60 μg/ml to 100 μg/ml, may lead to an improvement, but it does not lead to its complete eradication. The increase in ampicillin-resistant colonies was not linearly proportional to the increase in the number of bacteria added to the petri dish, probably due to the release of growth-inhibiting substances from cells killed by the antibiotic.
(18) Place the plate at room temperature until the liquid is absorbed.
(19) Invert the petri dish and incubate at 37°C. Colonies may appear after 12 to 16 h. The plate should be incubated at 37°C.