Experiments

(1) 150 mL of purified water was added to the medium powder (see figure below) and dissolved.

(2) Autoclaved at 121°C for 15 minutes.

(3) While autoclaving, the substances in the figure below were filter-sterilized.

(4) When the beaker after autoclaving reached about 70°C, the reagents sterilized in (3) were mixed.

(5) The material was poured into petri dishes and allowed to dry until it cooled and solidified.

(1)Medium powder (see figure below) was dissolved with 1 L of purified water.)

(2) Autoclave at 121°C for 15 minutes and cool to about 70°C.

(3) The material was poured into petri dishes and allowed to dry until it cooled and hardened.

(1) Centrifuge and remove the bacteria.

(2) Add 200 μL of DNAzol to 200 μL of bacteria, and suspend well by pipetting. Incubate for 2~5 min.

(3) Add 250 µL of 99.5% EtOH, and mix well by invert.

(4) After 3 min of incubation, centrifuge at 15,000 rpm for 2 min at 4°C, and remove the supernatant by decantation.

(5) The pellet was rinsed with 400 μL of 70% EtOH and centrifuged at 15,000 rpm at 4°C for 2 min.

(6) The supernatant was removed by decantation and centrifuged again at 15,000 rpm at 4°C for 1 min.

(7) The supernatant was completely removed by pipetting, and the lid was opened and allowed to dry for 1 min.

(8) The pellet was dissolved in 200 μL of 8 mMNaOH.

Sample Preparation

150 μl of DNA solution was prepared. If the volume is less than 150 μl, add water to make it 150 μl.

①Addition of Buffer

Add 450 μl of Binding Buffer DB to 150 μl of DNA solution, and mix by stirring strongly for 5 seconds.

②Adsorption on column

A NucleoSpin gDNA Clean-up Column (green ring) was set in a 2 ml collection tube. The solution of ① was added to the column and centrifuged at 11,000 × g for 30 seconds. The filtrate was discarded and the column was reassembled in the same tube.

③Membrane washing

First washing

Add 700 μl of Buffer DW to the column, agitate strongly for 2 seconds, and centrifuge at 11,000 × g for 30 seconds. The filtrate was discarded and the column was re-set in the same tube.

Second washing

Add 700 μl of Buffer DW to the column, agitate strongly for 2 seconds, and centrifuge at 11,000 × g for 30 seconds. The filtrate was discarded and the column was reassembled in the same tube.

④Membrane drying

The column was centrifuged at 11,000 × g for 1 minute. The column was placed in a microtube (1.5 ml) (prepared by each person).

⑤Elution of DNA

50 μl of Buffer DE was added to the column and allowed to stand for 1 minute without closing the lid, then the lid was closed and centrifuged at 11,000 × g for 30 seconds.

(1) Buffer QX1 was added in 3x volume (930 μL).

(2) Vortexed for 30s, and added QX2 (30 μL).

(3) Incubated at 50°C for 10 min (vortexed every 2 min) using a heat block (large).

(4) Centrifugation (13,500 rpm, rt, 1min) was used to remove the supernatant.

(5) 500 μL of BufferQX1 was added, followed by vortexing.

(6) Centrifugation (13,000, 1min, re) followed by removal of the supernatant.

(7) After adding 500 μL of BufferPE, vortexed.

(8) The pellet was air-dried for 40 min.

(9) 20 μL of MiliQ was added and vortexed or pipetted, followed by incubation at 50°C for 5 min.

(10) Centrifugation (13,000, 1min, re) was performed and the supernatant was transferred to a new tube.

(1) All amounts were appliqued to the column.

(2) Centrifugation (13,500 rpm, rt, 1min) was performed.

(3) Discard the flow-through and add 750 μL of PEbuffer.

(4) Centrifugation (13,500 rpm, rt, 1 min) was performed, and after discarding the flow-through, further centrifugation (13,500 rpm, rt, 1 min) was performed.

(5) The column was transferred to a new 1.5-mL tube and 10 μL of dH₂O was added.

(6) Incubated for 1min and centrifuged (13,500 rpm, rt, 1min).

(7) The amount of DNA was measured using Nano drop.

(1) About 2~5ml of E. coli bacteria solution containing plasmid was collected. ※Collection of about 2~3ml of bacteria was performed 2~3 times in a centrifuge (15000rpm, 1min.rt).

(2) The supernatant was discarded and centrifuged (15,000 rpm, 1 min, rt).

(3) The supernatant was completely removed using P200.

(4) 250 μl of Butter P1 (stored at 4°C) was added and subjected to a vortex mixer.

(5) Buffer P2 250 μl was added and mixed by vortex mixer 4~6 times.

(6) Add 350 μl of Buffer N3 and mix immediately by inverting 4~6 times.

(7) After centrifugation (13000 rpm, 10 min, rt), the supernatant was transferred to the attached column. ※⑤ to ⑦ were generally performed within 20 min.

(8) After centrifugation (13000rpm,1min,rt), the filtrate was discarded.

(9) Add 500 μl of Buffer PB and centrifuge (13000 rpm, 1min, rt).

(10) Discard the filtrate and add 750 μl of Buffer PE.

(11) After centrifugation (13000 rpm, 1min, room temperature), the filtrate was discarded.

(12) Centrifuge again (13,000 rpm, 1 min, room temperature).

(13) The column was placed in a new 1.5-ml tube, and 50 μl of Buffer EB was added so as not to contact the wall.

(14) The column was allowed to stand for 1 min.

(15) Centrifuged (13000 rpm, 1min., room temperature) and collected the concentrated liquid.

(16) The amount of extracted DNA was measured using Nanodrop.

(1) After adjusting the template DNA, agarose gel electrophoresis was always performed and the concentration was measured by comparison with a size marker of known concentration.

(2) After mixing the template DNA and analysis primers as follows, the mixture was adjusted to 14 μl.

(Ex) In case of plasmid DNA concentration of 100ng/ml and primer concentration of 10pmol/μl
Dispense 4 μl of DNA (400 ng) + 0.64 μl of primer (6.4 pmoles) + 9.36 μl of sterile water into 1 tube (total 14 μl)

(1) Tap water was filled to the level that the Sunoko (a disk with holes laid on the bottom) was slightly submerged.

(2) The apparatus and reagents in the basket were placed on top of it.

(3) The lid was set in the correct position and fixed by tightening the handle.

(4) The weight of the safety valve was checked to make sure that it was placed correctly, the exhaust cock was closed, the timer was set, and heating was started.

(5) As the temperature inside the equipment gradually increased, steam came out of the exhaust hose, but after a while the valve automatically closed and the internal pressure increased.

(6) After the set time passed, the temperature and internal pressure gradually decreased, and the exhaust valve opened at nearly 100°C.

(7) When the pressure became normal and the temperature dropped to below 100°C, the lid was opened and the instruments and reagents were removed after they cooled down enough to be held.

(1) To a DH5α competent cell (100µL), 2.0µL of plasmid solution (270.1ng/µL) was added.

(2) Incubated (on ice, 20 min).

(3) Incubate (42°C, 45sec).

(4) Incubated on ice for 2 min.

(5) 1mL of LB medium was added and suspended.

(6) Incubated (37°C, 1h).

(7) Centrifuge (13,000 rpm, 4°C, 1 min) and remove the supernatant.

(8) Suspended in 100 µL of LB medium.

(9) Two 10-fold dilution series were made.

(10) Spread onto LB+Cm (20 µM) plates.

(11) Incubated (37°C, overnight).

(1) Cells were harvested and counted.

(2) Cells were centrifuged and resuspended in electroporation reagent.

(3) Nucleic acid was added, and the mixture of nucleic acid and cells was transferred to a cuvette.

(4) The cells were transferred to a cuvette immediately after electroporation.

(5) The cells were incubated for 12 to 72 hours.

(6) Assay was performed.

(1) Preparation of reaction solutionThe composition of the solution is shown below.

(2) ncubated at 37°C for 5-15 min.

(1) Gel preparation: The concentration of the gel to be used was determined according to the length of the target DNA.

(2) Sample preparation: DNA samples and other samples to be investigated were diluted with appropriate solvents. For dilution of samples, appropriate solvents were selected according to the desired concentration and reaction conditions. The choice of solvent was carefully determined based on the purpose of the experiment and the detection method to be used.

(3) Loading samples onto gels: Samples prepared on gel cast trays were injected into the holes. Samples were loaded precisely using a micropipette or micropipette. Care should be taken during sample loading to prevent cross contamination. Point-like loading and accurate positioning of samples are also important.

(4) Performing electrophoresis: The gel cast tray was set into the electrophoresis bath and TAE buffer was poured into the bath. Electrophoresis was conducted at the appropriate power and time using a power supply device with electrodes connected to the electrophoresis tank. The conditions of electrophoresis (voltage and time) should be optimized according to the size and purpose of the sample.

(5) Visualization of bands: Once electrophoresis was complete, the gels were stained with MIDORIGREEN. The staining allowed DNA fragments to be visualized as bands. After staining, the gel was photographed and the position and intensity of the bands were recorded.

(6) Analysis of results: The position and size of the stained bands were analyzed to obtain the desired information. Gel image analysis software or dedicated analysis tools can be used for the analysis. The analysis results were accurately recorded and necessary statistical analysis and graphing were performed.

(1) Measured in milli-Q water.

(2) Blank was measured in buffer.

(3) Concentration of the solution with plasmid was measured.

A 50% glycerol solution was prepared by diluting 100% glycerol and MiliQ in a 1:1 ratio. The solution was then autoclaved.

Bacteria were cultured overnight in appropriate culture medium (e.g., LB medium).

500 µL of the cultured bacterial stock was taken and mixed with 500 µL of 50% glycerol solution.

This mixture was placed in a sterile 2 ml tube.

Freeze the tubes at -80°C.

The tubes were frozen at -80°C.

Glycerol stock can be stored stably at -80°C for a long period of time, but repeated cycles of freezing and thawing will degrade it, so avoid as much as possible.

Scribe the contents with an oil-based pen so that the contents can be identified.