Team:TUST China/Protocol
Experiment Protocol
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Tryptone 10 Yeast Extract 5 Sodium Chloride 10 Add 5mol／L Sodium hydroxide, until its pH reach to 7.0~7.4 Add ddH2O to 1L Sterilization: Autoclave 120℃ for 20min
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Tryptone 10 Yeast Extract 5 Sodium Chloride 10 Add 5mol／L Sodium hydroxide, until its pH reach to 7.0~7.4 Add ddH2O to 1L Sterilization: Autoclave 120℃ for 20min
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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).
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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.
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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.
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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.
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Cell: chemically-competent E.coli cell (stored at -80℃)
Agent: SOC culture media.
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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.
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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.
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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.
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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)
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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.
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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℃.
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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.
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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
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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.
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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
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Components Volume(µL) PrimeSTAR Mix 75 FW Primer 5 RV Primer 5 ddH2O 64 Template 2 Total volume 150 µL Operation Process:
1. Prepare the mix for all the samples in a single 1.5mL tube.
2. Add the total volume per sample (without DNA) to each PCR tube.
3. Add the template DNA to each tube.
4. Put the tubes in the PCR machine and apply the corresponding program.
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1. After the formation of gel, pull out the gel comb and take the gel out of the mould and then add 6 µL samples containing 1µL loading buffer into gel pores.
2. Immerge the gel with 1×TAE buffer in the electrophoresis chamber.
3. Using pipette to add marker and samples to different pore. (The content of pore depends on the gel comb, there are 3 kinds of volume, 10μL, 20μL, and 50μL) Do not stick the bottom and side of gel pore to prevent the leakage.
4. Turn on the electrical source to start the electrophoresis, the voltage is set at 120-150V and the electrophoresis time is set at 30min.
5. After the electrophoresis process end, the gel is observed under blue light or ultraviolet.
6. Cut the DNA fragment from agarose gel under 365nm ultraviolet with a clean, sharp scalpel. (Only for recycling the DNA products.)
7. Weigh the gel, measure its concentration and slice in recycling box.
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(This protocol uses the commercially available TIANprep Column DNA Gel Extraction Kit from TIANGEN BIOTECH (BEIJING) CO., LTD. We have made some adaptation and for detailed user-guide and list of required reagent, see TIANprep Column DNA Gel Extraction Kit handbook.)
1. Add 500 μl Buffer BL to Spin Columns CA2, centrifuge at 12000 rpm for 1 min. Discard the flow-through and place the Spin Column back into the collection tube.
2. Cut the DNA fragment from agarose gel, place them into the clean centrifuge tube and weigh the gel.
3. Add an equal volume Buffer PN to the gel and incubate at 50°C water bath. During the process, gently turn it over and down until the agarose gel dissolves completely.
4. Transfer the solution collected into the Spin Columns CA2, incubate at room temperature for 2 min and centrifuge at 12000 rpm for 30~60s. Discard the flow-through and place the Spin Column back into the collection tube.
Note: The volume of Spin Column is 800 μl, so the sample can be added in batches if the volume of sample is larger than 800 μl.5. Add 600 μl Buffer PW to the Spin Columns CA2, centrifuge at 12000 rpm for 30~60 s. Discard the flow-through and place the Spin Column back into the collection tube.
6. Repeat step 5.
7. Place the Spin Column CA2 back into the clean collection tube, centrifuge at 12000 rpm for 2 min and remove residual Buffer PW. Place Spin column CA2 at room temperature for several minutes and dry it out to prevent residual Buffer PW affecting the next experiment.
Note: the residual ethyl in Buffer PW will affect the enzyme reaction in the subsequent steps.8. Transfer the Spin Column CA2 to a clean centrifuge tube. Add Elution Buffer to the center of the membrane, incubate at room temperature for 2 min, then centrifuge at 12000 rpm for 2 min. Store the DNA solution properly.
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(This protocol uses the commercially available TIANprep Column DNA Gel Extraction Kit.from TIANGEN BIOTECH (BEIJING) CO., LTD. We have made some adaptation and for detailed user-guide and list of required reagent, see TIANprep Mini Plasmid Kit handbook.)
Preparation: add ethyl into Buffer PW, the volume referring to the label on the bottle.
1. Add 500 μl Buffer BL to Spin Columns CP3, centrifuge at 8000 rpm for 1 min. Discard the flow-through and place the Spin Column back into the collection tube.
2. Add 1~5 ml samples to the centrifuge tube, centrifuge at 8000 rpm for 1 min, discard the supernatant.
3. Add 250 μl Buffer P1 to the centrifuge tube with bacterial sediment, suspend the sediment.
Note: It will affect the lysis, resulting in lowering the quantity of extraction and its purity, if there are unevenly mixed bacteria.4. Add 250 μl Buffer P2 to the centrifuge tube, gently turn it over and down 6~8 times to split bacteria completely.
5. Add 350 μl Buffer P3 to the centrifuge tube, gently turn it over and down 6~8 times right away, mix evenly and white floc will appear at this time.
Note: In order to avoid local precipitation, it should be mixed immediately after Buffer P3 is added.6. Transfer the supernatant collected into the Spin Columns CP3, centrifuge at 12000 rpm for 30~60 s. Discard the flow-through and place the Spin Column back into the collection tube.
7. Alternative: Add 500 μl Buffer PD to the Spin Columns CP3, centrifuge at 12000 rpm for 30~60 s. Discard the flow-through and place the Spin Column back into the collection tube.
8. Add 600 μl Buffer PW to the Spin Columns CP3, centrifuge at 12000 rpm for 30~60 s. Discard the flow-through and place the Spin Column back into the collection tube.
9. Repeat step 8.
10. Place the Spin Column back to the collection tube, centrifuge at 12000 rpm for 2 min and remove residual Buffer PW.
11. Transfer the Spin Column to a clean centrifuge tube. Add 50~100 μl volume of Elution Buffer to the center of the membrane, incubate at room temperature for 2 min and centrifuge at 12000 rpm for 2 min, then collect plasmid solution to the centrifuge tube.
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1.Prepare the mix for all the samples in a single 1.5mL tube (mind pipetting error!). For one sample:
Components Volume(µL) Taq Mastermix 2X 75 FW Primer 5 RV Primer 5 ddH2O 64 Template 2 Total volume 150 µL 2. Pipette 15µL of mix into each PCR tube (one per colony).
3. Under sterile conditions, pick a colony into the surface of corresponding petri dish and then incubate into each PCR tube.
4. Put the tubes in the PCR machine and apply the following program (for different sets of primers the annealing temperature needs to be adjusted):
Step Temperature (ºC) Time Operation Initial denaturation 95 2min Denaturation 95 30ec x30 cycles Annealing 55-65(choose one ) 30sec Extension 72 1.5min/1kb Final extension 72 5min Holding Temperature 22 hold 5. While the PCR is running, Cast an agarose gel following the DNA electrophoresis protocol.
6. Once the PCR program is finished, transfer 6µL of PCR product into the gel pores containing the loading buffer and follow the DNA electrophoresis protocol.
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1. Cut DNA fragments and vectors at specific sites with Restriction Endonuclease, recycle the agarose gel electrophoresis products
2. Measure the concentrations of DNA fragment and vector
3. Add proper ddH2O, DNA fragments and vectors to the tube in order, suspend solution (5 μL) to mix up evenly
4. Alternative: Incubate at 65℃ for 3min, undergo ice bath immediately
5. Add 5 μL Solution I to the connection system, suspend solution to mix up evenly and centrifuge briefly
6. Label the tube and being unsealed, place it at 16℃ water bath for 1h.
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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.)
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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.
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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.
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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)
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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
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