Experiments with the Hanahan method for the preparation and transformation of susceptible Escherichia coli (efficient transformation strategies)
Experiments with the Hanahan method for the preparation and transformation of susceptible Escherichia coli (efficient transformation strategies)
In the late 1970s and early 1980s, when Dong Hanahan was a graduate student at Cold Spring Harbor Laboratory and Harvard University, he made previously unheard of transformation efficiencies, and later standardized his methods. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Experiments with the Hanahan method for the preparation and transformation of susceptible Escherichia coli (efficient transformation strategies)
Principle
While Dong Hanahan was a graduate student at Cold Spring Harbor Laboratory and Harvard University in the late 1970s and early 1980s, he made previously unheard-of conversion efficiencies, and later standardized his methods.
Materials and Instruments
Plasmid DNA E. coli Move I. Materials For more product details, please visit Aladdin Scientific website.
Methyl sulfoxide DMSO DnD solution 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) Methyl sulfoxide DMSO
The oxidation product of dimethyl sulfoxide is presumed to be dimethyl sulfide, an inhibitor of conversion.
(2) DnD solution (DMSO and DTT)
DTT (dithiothreitol) 1.53 g, DMSO 9 ml, 1 mol/L potassium acetate (pH 7.5) 100 μl, H2O made up to 10 ml.
The DnD solution was filtered and sterilized with organic solvent resistant Millex SR membrane (Millipore). 160 μl of DnD solution was dispensed into 0.5 ml sterile microcentrifuge tubes, sealed, and stored at -20℃.
(3) Transformation buffer
Standard Transformation Buffer (TFB) is usually prepared on the spot. Freezing buffer (FSB) is used to freeze (-70℃) the sensory cells.
2. Culture medium
(1) SOB Agar Plate containing 20 mmoI/L MgSO4 and appropriate amount of antimicrobials
Standard SOB agar plates contain 10 mmoI/L MgSO4.
(2) SOB medium with 20 mmol/L MgSO4
Standard SOB medium contains 10 mmol/L MgSO4.
(3) SOC medium
1 ml of SOC medium is required for each transformation reaction.
3. Nucleic acids and oligonucleotides
Plasmid DNA (recombinant plasmid)
4. centrifuges and rotors
Sorvall GSA turntable or equivalent
5. Specialized equipment
(1) Liquid nitrogen
(2) Pre-cooled polypropylene centrifuge tubes (50 ml)
(3) Pre-cooled polypropylene centrifuge tubes (17x100 mm; Falcom 2059)
(4) Pre-set 42°C water baths
6. Carriers and strains
Frozen E. coli for transformation
The strain should be stored at -70℃.
Methods
1. Cell preparation
(1) Preparation of transformation buffer
TFB can be used for the preparation of ready-to-use perplexed recipient cells, and FSB can be used for the preparation of receptor cells stored at -70°C. Organic contamination of the water from which the Transformation Buffer is prepared will reduce the rate of transformation of the receptor cells. Therefore, using water obtained directly from a good quality Milli-Q filtration system (Millipore) will give satisfactory results. In case of problems, deionized water can be treated with activated carbon prior to use.
① Preparation of Standard Transformation Buffer
A. Preparation of 1mol/L MES buffer: Dissolve 19.52 g of MES in 80 ml of pure water (Milli-Q grade, or equivalent), adjust the pH to 6.3 with 5 mol/L KOH, and finally dilute to 100 ml with purified water, and then filter through a pre-treated Nalgene membrane (0.45 μm pore size) to remove bacteria. Dispense in 10 ml portions and store at -20℃.
B. Preparation of TBF solution: Dissolve the ingredients listed in the table below in 500 ml of water, then add 10 ml of 1 mol/L MES buffer (pH 6.3). Finally, make up to 1 L with purified water. 
C. The TFB solution was filtered through a pre-treated NaIgene filter (0.45 μm pore size) to remove bacteria, stored in 40 ml portions in tissue culture flasks (Corning, or equivalent) and stored at 4°C. The TFB solution was then filtered through a NaIgene filter (0.45 μm pore size).
Preparation of freezing buffer
A. Dissolve 9.82 g of potassium acetate in 90 ml of pure water (Milli-Q grade or equivalent) to make 1 mol/L potassium acetate, then adjust the pH value to 7.5 with 2 mol/L acetic acid, add purified water to the final volume of 100 ml, divide the solution into small portions, and store at -20℃.
B. Preparation of FSB solution: use 500 ml of pure water to dissolve all the ingredients listed in the table below, wait until the reagents are dissolved, use 0.1 mol/L HCl to adjust the pH value to 6.4. If over-adjusted, do not use alkali to re-adjustment, but should be discarded to re-make the solution. Make up the volume to 1 L with purified water.
C. The solution is sterilized by filtration through a pre-treated Nalgene membrane (0.45 μm pore size). Dispense into 40 ml portions and store in tissue culture flasks (Corning or equivalent) at 4°C. During storage, the pH of the solution will decrease to 6.1-6.2, after which it will stabilize.
(2) Line the surface of SOB agar plates with the desired strain directly from the frozen E. coli storage solution using a sterile inoculating loop and incubate at 37°C for 16 h. The plate will be incubated for 16 h at 37°C with a sterile inoculating loop.
It is not necessary to thaw the frozen bacteria; the bacteria carried by the inoculating loop across the surface of the frozen bacteria are sufficient. One tube of frozen bacteria can be used more than once.
(3) Transfer 4~5 well-separated colonies to 1 ml of SOB containing 20 mmol/L MgSO4 . Shake at medium speed to disperse the bacteria, and then dilute the culture with 30-100 ml of SOB containing 20 mmol/L MgSO4 in a 1 L conical flask.
(4) Incubate the bacteria at 37℃ for 2.5~3 h and monitor the growth of the culture.
For efficient transformation, the number of viable cells must be less than 108 cells/ml, which corresponds to an OD600 value of about 0.4 for most E. coli. To ensure that the growth density of the bacterial culture is not too high, the OD600 value can be measured at 15 - 20 min intervals to monitor the growth, and the time of the measurement and the OD600 value can be used to make a graph to predict the incubation time for the culture to reach an OD600 value of 0.4, and the bacterial culture can be collected when the OD600 value reaches 0.35.
For unspecified reasons, there are two periods in the growth curve of E. coli that can have higher transformation efficiency, one at the beginning ( OD600 =0.4) ( Hunahan 1983) and the other at the end ( OD600=0.95) ( Tang et al, 1994). The easy effectiveness of the bacteria harvested at the early stage is due to the fact that its high transformation rate can be sustained for a longer period of time, whereas at the late stage, the growth peak is steeper, and a slight delay of 2 to 3 min in collecting the bacteria will reduce the transformation efficiency by an order of magnitude.
The relationship between OD600 values and the number of viable cells per milliliter varies greatly from strain to strain, so it was necessary to standardize the spectrophotometer readings by measuring the OD600 values of specific E. coli growth cultures at different phases of the growth cycle and spreading each dilute concentration of culture on antibiotic-free LB agar plates to calculate the number of viable cells at each phase.
(5) Transfer the bacteria into a sterile, single-use, ice-pre-chilled 50 ml polypropylene tube and leave on ice for 10 min to allow the culture to cool to 0 °C.
(6) The cells were recovered by centrifugation at 2700 g (equivalent to 4100 r/min with Sorvall's turntable) for 10 min at 4 °C.
(7) Pour out the culture medium and invert the tube for 1 min to allow the last remaining trace of culture medium to drain.
(8) Resuspend the precipitate with approximately 20 ml (per 50 ml tube) of ice-cold Transformation Buffer (TFB or FSB) and place the resuspended cells in an ice bath for 10 min.
(9) Recover the cells by centrifugation at 2700 g (equivalent to 4100 r/min with Sorvall's turntable) for 10 min at 4 °C.
(10) Pour out the culture solution and invert the tube for 1 min to allow the last remaining trace of culture solution to drain.
(11) Resuspend the precipitate with 4 ml (per 50 ml tube) of ice-cold Transformation Buffer (TFB or FSB) by gentle shaking. Prepare the sensory cells for immediate use according to the procedure given in step (12) Part I, and the sensory cells stored at -70°C for later use according to step (12) Part II.
2. Preparation of sensory cells
(12) Preparation of Transformed Receptor Cells
① Preparation of Fresh Receptor Cells
A. Add 140 μl of DnD solution to the center of each cell suspension, immediately swirl gently to mix the suspension, and then place on ice for 15 min.
B. Add another 140 μl of DnD solution to each tube, gently swirl to homogenize the suspension, and place on ice for 15 min.
C. Dispense small portions of the suspension into cooled sterile polypropylene tubes (17x100 mm) and place the tubes on ice.
② Preparation of frozen sensory cells
A. Add 140 μl of DMSO to every 4 ml of resuspended cell solution, gently rotate to mix, and place the suspension on ice for 15 min.
B. Add 140 μl of DMSO to each cell suspension, gently rotate and mix well, and put the suspension on ice for 15 min.
C. Quickly dispense the suspension into cooled sterile microcentrifuge tubes or tissue culture tubes, seal the tubes tightly, and freeze the sensory cells in liquid nitrogen. Store at -70℃ for preparation.
D. When needed, remove a tube of sensory cells from the -70℃ 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 on ice for 10 min.
E. Using a cold sterile pipette tip, transfer the sensory cells to a cooled sterile polypropylene tube (17 x 170 mm) and place on an ice bath.
3. Transformation
Include positive and negative controls.
(13). Add the DNA fragments to be transformed to the tube containing the sensory cells (25 ng of DNA for 50/4 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 two control tubes in the experiment: one containing 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.
(14) Place the tubes in circulating water pre-warmed to 42°C and leave for exactly 90 s without shaking the tubes.
Thermal stimulation is a critical step and it is very important to reach the thermal stimulation temperature accurately. The temperatures and incubation times shown here are for measurements using Falcon 2059 tubes; other types of tubes may give different results.
(15) Quickly transfer the tubes to an ice bath and allow the cells to cool for 1~2 min.
(16) Add 800 μl of SOC medium to each tube and 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 bacteria, 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 appearance of satellite colonies sensitive to cyanobenzylpenicillin. The use of carboxybenzylpenicillin instead of ampicillin in the selection medium, as well as an increase in the antibiotic concentration from 60 μg/ml to 100 μg/ml, may lead to an improvement but not 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 for 12 hours.