Strain: DH5 alpha (stored in glycerol tube at -80℃)

Agents: LB broth culture, glycerol, Calcium chloride

Devices: three triangular flasks, a centrifugal cup (50ml), two shake tubes,

a EP tube (10ml) and fifty EP tubes (1.5ml).

2.1 Shake tube, triangular flask, centrifuge cup, rinse with ddH2O repeatedly without any cleaning agents. If cleaning agent is used, a lot of ddH2O should be used to rinse cleaners. It is really important that the cleaning agent will seriously devastate the transformation efficiency of the chemically-competent cell.

2.2 All the devices dried in an oven at 50~100 ℃ after sterilization for 15 min at 121℃ (0.1 MPa)

2.3 Prepare LB medium 100 mL, packed separately, two shake tubes each 5 mL and a triangular flask of 50 mL, sterilized in autoclave at 121℃ (0.1 MPa) for 15 min.

2.4 Prepare 20 mL Calcium chloride solution (0.1 M), filtered in triangle bottle.

3.1. Inoculate a loop of fresh single-colony E.coli onto 25 mL LB broth culture in 100 mL triangular flask. Incubate at 180 r/min, at 37℃ in shaker overnight.

3.2. Inoculate 1mL seed culture solution into 100 mL LB broth culture media in 500 mL triangular flask. Incubate 180 r/min, 37 ℃ in shaker until OD600 reach to 0.4-0.5

3.3. Transfer the incubated solution to two 50 mL centrifugal tube respectively. Put these on ice for 30 min. Centrifuge 4000 r/min for 10 min at 4℃ . Discard the supernatant.

3.4. Add 10 mL pre-cool calcium chloride and re-suspend the solution. Centrifuge 4000r/min for 10 min at 4℃ . Discard the supernatant.

3.5 Repeat step 4

3.6 Add 5 mL pre-cool calcium chloride - glycerin into the centrifugal tube, re-suspend and transfer 100 μL to 1.5 mL sterilized EP tube. Thus the chemically competent cell is ready.

4.1. Choose a single colony from the newly activated plate and avoid using plates placed at 4℃ for a long time.

4.2 The OD600 of the bacteria should be kept within the range of 0.2~0.3, preferably no more than 0.4.

4.3 In the production process, it is necessary to ensure that all solutions and containers are pre-cool and the bacteria are always on ice.

Cell: chemically-competent E.coli cell (stored at -80℃)

Agent: SOC culture media.

2.1 Shake tube, triangular flask, centrifuge cup, rinse with ddH2O repeatedly without any cleaning agents. If cleaning agent is used, a lot of ddH2O should be used to rinse cleaners. It is really important that the cleanin g agent will seriously devastate the transformation efficiency of the chemically-competent cell.

2.2 All the devices dried in an oven at 50~100 ℃ after sterilization for 15 min at 121℃ (0.1 MPa)

2.3 Prepare LB medium 100 mL, packed separately, two shake tubes each 5 mL and a triangular flask of 50 mL, sterilized in autoclave at 121℃ (0.1 MPa) for 15 min.

2.4 Prepare 20 mL Calcium chloride solution (0.1 M), filtered in triangle bottle.

3.1. Inoculate a loop of fresh single-colony E.coli onto 25 mL LB broth culture in 100 mL triangular flask. Incubate at 180 r/min, at 37℃ in shaker overnight.

3.2. Inoculate 1mL seed culture solution into 100 mL LB broth culture media in 500 mL triangular flask. Incubate 180 r/min, 37 ℃ in shaker until OD600 reach to 0.4-0.5

3.3. Transfer the incubated solution to two 50 mL centrifugal tube respectively. Put these on ice for 30 min. Centrifuge 4000 r/min for 10 min at 4℃ . Discard the supernatant.

3.4. Add 10 mL pre-cool calcium chloride and re-suspend the solution. Centrifuge 4000r/min for 10 min at 4℃ . Discard the supernatant.

3.5 Repeat step 4

3.6 Add 5 mL pre-cool calcium chloride - glycerin into the centrifugal tube, re-suspend and transfer 100 μL to 1.5 mL sterilized EP tube. Thus the chemically competent cell is ready.

4.1. Choose a single colony from the newly activated plate and avoid using plates placed at 4℃ for a long time.

4.2 The OD600 of the bacteria should be kept within the range of 0.2~0.3, preferably no more than 0.4.

4.3 In the production process, it is necessary to ensure that all solutions and containers are pre-cool and the bacteria are always on ice.

Two shake tubes, a triangular flask, a centrifugal cup (50 ml), fifty EP tubes (1.5 ml).

Two YT culture medium(1.6% tryptone, 1% yeast extract and 0.5% sodium chloride)

2.1 Shake tube, triangular flask, centrifuge cup, rinse with H2O repeatedly without any cleaning agents. If cleaning agent is used, a lot of water should be used to rinse cleaners. It is really important that the cleaning agent will seriously devastate the transformation efficiency of the cell. Finally, rinse with ddH2O twice.

2.2 All the devices dried in an oven at 50~100 ℃ after sterilization for 15 min at 121℃ (0.1 MPa).

2.3 Prepare two YT medium 150 mL, packed separately, two shake tubes each 5mL and a triangular flask of 100 mL, sterilized in autoclave at 121℃ (0.1MPa) for 15 min.

2.4 Prepare 200 ml 10% glycerol, sterilized in autoclave at 121℃ (0.1 MPa) for 15 min.

2.5 Prepare 200 ml ddH2O, sterilized in autoclave at 121℃ (0.1 MPa) for 15 min.

3.1 Inoculate a loop of DH5 alpha onto LB broth culture. Incubate at 37℃ in shaker overnight.

3.2 Inoculate single-colony on plate into shake tubes. Incubate at 37℃ in shaker overnight.

3.3 Inoculate 1% seed culture solution into shake bottle. Incubate 200 r/min, 37 ℃ in shaker. 2.5 h after Inoculation, start to measure the figure of OD600.

3.4 Put the shake bottle on ice for 10 min when OD600 reach to 0.4~0.5. At the same time. Pre-cool the ddH2O, 10% glycerol and the centrifugal cup (50 ml).

3.5 Transfer the incubated solution to 50 mL centrifugal cup. Centrifuge 5000 r/min for 5 min at 4℃

3.6 Discard the supernatant, add 5 ml pre-cool ddH2O approximately, suspend the solution, and then, add about 40 ml pre-cool ddH2O. Centrifuge 5000 r/min for 5 min at 4℃, repeat twice.

3.7 Discard the supernatant, add 5 ml pre-cool 10% glycerol approximately, suspend the solution, and then, add about 40 ml pre-cool 10% glyc-erol. Centrifuge 5000 r/min for 5 min at 4℃, repeat twice. In the meanwhile, pre-cool 1.5 ml EP tube.

3.8 Discard the supernatant, add 500 μL pre-cool 10% glycerol, suspend the solution. Packed separately to 1.5 ml EP tubes (each 50 μL).

3.9 The last is used to measure contamination.

3.10 Pack all the competent cells to sample bags, sign and stored at -80℃.

4.1 Choose a single colony from the newly activated plate and avoid using plates placed at 4℃ for a long time.

4.2 The OD600 of the bacteria should be kept within the range of 0.6~0.8, preferably no more than 0.8.

4.3 In the production process, it is necessary to ensure that all solutions and containers are pre-cool and the bacteria are always on ice.

4.4 If the efficiency can not meet the experimental requirements, SOB medium can be used to replace 2 x YT medium. SOB medium: 2% peptone, 0.5% yeast powder, 0.05% sodium chloride, 2.5 mM Potassium chloride; the final concentration of magnesium chloride is 10 mM，packed separately and added along.

4.5 In every step, wash and remove the supernatant as quickly as possible after centrifugation. Otherwise, bacteria at the bottom of the tube will become loose, resulting in a large loss in discard.

Cell: Electrocompetent cell of E.coli (stored at -80℃)

Agent: SOC culture media，ethyl

Devices: Gene Pulser and Pulse Controller apparatus for electrophoration Filter Paper

2.1. Wash the device: add ethyl to the pulser for cleaning the interior

2.2Put the pulser on ice for 10min and heat the SOC culture media at 37℃

2.3 Chill 100 μL prepared cell on ice for 10 minutes

2.4. Add 0.1~10 ng plasmid DNA to the cell, mix up evenly and keep it on ice for 10~30min

2.5. Transfer in to the pulser in the ice-cold state

2.6. Electroporation Procedure

2.7. Incubate at 180 r/min in shaker for retrieving

2.8. Wash the pulser with ddH2O and store in ethyl

2.9. Centrifuge at 8000 rpm for 2min, discard supernatant, suspend 200 μL bacterial cells and plate bacteria onto agar plates with ampicillin, incubate under favorable conditions for 12h at least.

3.1. Clean the interior of pulser out

3.2. Remain samples in ice-cold state can enhance the transformation efficiency.

3.3. Pre-cool pulser is absolutely necessary and significant

3.4. be carefully and gently to mix up the sample as the call is really fragile after electroporation

3.5. Resuscitation time can be prolonged, but no more than 2h

Aquaculture, fish pond water samples and stool samples. (From the area with high tetracycline content. Water samples need to be protected from light, and the following operations are required to avoid light.)

1. Centrifuge the water sample in the 50ml centrifuge tube（8000-10000r/min, 5min）and take the supernatant in the clean centrifuge tube. Repeat many times until there is no visible impurity. (Repeat this procedure no less than three times.)

2. The water sample in the centrifugal tube is passed through the membrane and packed into 10 clean centrifugal tubes, each containing 10 ml.

3. Placing 10 centrifuge tubes at -80 degrees Celsius for no less than 24 hours.

4. The samples were sealed with preservative film after 24 hours of freezing, and several small holes were punctured. The samples were lyophilized by freeze-drying machine until they were all turned into powder.

1. Take appropriate amount of fecal samples in 50 ml centrifugal tube with water solution, centrifugal(8000-10000r/min，5min）, take supernatant in a clean centrifugal tube. Adding water to dissolve, centrifugation （8000-10000r/min，5min）, and take the supernatant in centrifugation. (Repeated operation above, all the supernatant was placed in centrifuge tube.

2. The supernatant will be centrifugally repeated several times until there is no visible impurity.

3. The samples in the centrifugal tube were passed through the mmbrane and packed into 10 clean centrifugal tubes, each containing 10 ml.

4. Placing 10 centrifuge tubes at -80 degrees Celsius for less than 24 hours.

5. Freezing the sample after 24 hours with a plastic wrap and seal a plurality of small holes.

6. The lyophilized machine was used for freeze-drying operations until all the samples were changed into powder.

1. The freeze-dried powder is merged into a centrifugal tube, dissolved in a small amount of water (just dissolved, water cannot be excessive), and put in - 80 degrees Celsius freezing 24 hours, freeze-dried.

2. Adding 1mlDMF to dissolve the powder (over film treatment) and get the final samples. (Need to operate in hood)

1. Instrument: WatersC18ODS-3 column (250 mm 4.6 mm, 5 m).

2. Reagents:

Mobile phase A solution: methanol (filtration treatment) B liquid: ddH2O

Note:

Ultrasound is needed for at least half an hour before use and the temperature should not be too high. Injection needles need to be cleaned by mobile phase. Use brown liquid vial

3. Setting speed: 1 mL/minColumn

temperature: 35 degrees Celsius

Absorption peak: 355nm

Injection volume: 20ul

Setting Concentration Gradient：

Time	A	B
0-2min	5%	95%
2.1min	40%	60%
8min	70%	30%
8.1-12min	90%	10%
12-15min	5％	95%

This method should peak at about 6.72min.

Please keep all the data and images of the liquid phase and send the results to TUST_China

  tustigem2018@163.com