Participants

Goal

• Prepare LB medium (1L) for bacterial culture
• Prepare 100mg/ml Ampicillin
• Prepare 0.05mol/L CaCl2
• Inoculate Azurin template strain, anti-PD-L1 template strain, plactate1 template strain, plactate2 template strain

Content

Participants

Goal

• Plasmid extraction
• PCR amplification
• Use agarose gel electrophoresis to detect whether DNA was obtained
• DNA recovery

Content

• Plasmid was extracted：
  

  Material	Dosage
  Solution I: SP1 bacterial suspension	250μL
  Solution II: SP2 cell lysis buffer	250μL
  Solution III: SP3 neutralization solution	350μL
  Wash solution	750μL

  1-5ml of bacterial culture (P1, P2, Azurin, anti-PD-L1) was centrifuged at 12000rpm for 1min, and the supernatant was absorbed
  • Add 250μL solution to the centrifugation tube containing the bacterial precipitation and suspend the bacterial cell precipitation thoroughly using a vortex oscillator
  • Add 250μL solution Ⅱ into the centrifuge tube and gently turn it up and down 6-8 times to fully crack the bacteria. At this time, the bacteria solution will become clear and viscous
  • Add 350u solution III into the centrifuge tube and turn it up 6-8 times to mix well. The white, fluffy precipitate will appear at this time. Centrifuge at 12000rpm for 10min and transfer the supernatant to another clean centrifuge tube
  • Add 0.35 times the volume of anhydrous ethanol and mix well
  • Add the supernatant into the adsorption column, leave at room temperature for 2min, centrifuge at 12000rpm for 1min, drain the waste liquid in the collection tube, and put the adsorption column back into the collection tube
  • Add 750μL bleach solution to the adsorption column, centrifuge at 12000rpm for 1min, and put the adsorption column into the collection tube
  • Add 700L bleaching solution to the adsorption column, centrifuge at 12000rpm for 1min, and put the adsorption column into the collection tube
  • Centrifuge at 12000rpm for 2min and place the adsorption column at room temperature or 50℃ for several minutes
  • Place the adsorption column into a clean centrifuge tube, drop 50μL eluent preheated in a water bath at 65℃ into the center of the adsorption film, place at room temperature for 2min, and centrifuge at 12000rpm for 1min
  • The eluent obtained was re-added into the adsorption column, placed at room temperature for 2min, and centrifuged at 12000rpm for 1min
• PCR amplification (target gene amplification)：
  

  Material	Dosage
  Target gene (P1, P2, anti-PD-L1, Azurin)	2 μL
  2*Mix (dNTP, DNA polymerase, Buffer, magnesium ion)	25 μL
  Primer (F)	2 μL
  Primer (R)	2 μL
  ddH2O	19 μL

  • Set up the PCR reaction program
  • Set up the PCR reaction program
  Program:
  
  1.Pre degeneration： Double chain to single chain 95°C，5min
  2.dimorphism:95°C，30s
  3.annealing：55°C，30s （Repeat 1-3 30 times）
  4. extending：72°C，30s for gene /2min for plasmid
  5. 72 ℃，10 min
  6. 4°C，∞
  • Initiate the program
  • Agarose gel electrophoresis was used to detect whether DNA was amplified
  • Prepare50mL agarose gel

  Material	Dosage
  Agarose powder	0.5 g
  TAE buffer solution	50 mL
  Nucleic acid dye	3 μL

  • Melt agarose powder and TAE buffer at high temperature and add 3μL nucleic acid dye
    
  • Install the electrophoresis tank
  • Gently pour the electrophoresis solution cooled to 60 °C onto the horizontal plate of the electrophoresis tank
  • After it has solidified, add the electrophoresis buffer to the bath and pull out the comb
  • Add the mixture to the sample tank
  • Connect one end of the sample hole to the negative electrode and the other to the positive electrode. Turn on the power supply, adjust the voltage to 180V, and electrophoresis for 20min
  • The gel was removed and observed under an ultraviolet lamp. The DNA bands with red fluorescence were found
  
  
• DNA gel extraction
  

  Material	Dosage
  Glue solution	300 μL / 0.1 g DNA gel block
  Bleach wash solution	600 μL

  • After agar-gel electrophoresis, a single DNA strip of interest is cut from the agar-gel and weighed
  • Add 600μL sol solution to the glue block and place in a water bath at 55℃ for 10 min. During the process, gently turn the centrifuge tube up and down continuously to ensure that the glue block is fully dissolved
  • The solution was added to an adsorption column and centrifuged at 12000pm for 60 seconds
  • Add 600μL bleaching solution to the adsorption column and centrifuge at 12000rpm for 1min, discard the waste liquid, and put the adsorption column into the collection tube
  • Add 600μL bleach solution into the adsorption column, centrifuge 12000rpm for 1min, discard the waste liquid, and put the adsorption column into the collection tube
  • Centrifuge at 12000rpm for 2min, remove the bleach solution as much as possible, and place the adsorption column open at room temperature or 50℃ in a temperature box
  • Place the adsorption column into a clean centrifuge tube, drop the appropriate amount of eluent preheated in a water bath at 65℃ into the center of the adsorption film, place at room temperature for 2min, and centrifuge at 12000rpm for l min
  • DNA products stored at -20℃

Result

Participants

Goal

• The target gene sfGFP was obtained by PCR
• Recombinant plasmid was constructed by homologous recombination
• The recombinant product transformed Escherichia coli BL21

Content

• The target gene sfGFP was obtained by PCR
  PCR system
  Target gene (P1, P2, sfGFP): 2μL
  2*Mix (dNTP, DNA polymerase, Buffer, magnesium ion): 25 uL
  Primer (F/R): F:2μL R:2μL
  ddH2O: 19μL set the PCR reaction program
  Procedure: Pre degeneration: double chain to single chain 95°C，5min
  Degeneration：95°C，30s
  Annealing：55°C，30s （Repeat 30 times）
  Extending：72°C，30s for GFP /2min for plasmid
  72 ℃，10 min
  4°C，∞
  agarose gel formula
  

  Material	Dosage
  Total	50 mL
  Agarose	0.5 g
  1*TAE	50 mL

  After 2 minutes of high-fire melting, add 3μL of dye and pour it into the mold. 
  
• Homologous recombination
  the amount of DNA required for the recombination reaction was calculated according to the formula, and the amount of each component added was not less than 1μl
  Prepare the following reaction system on the ice:
  

  Component	Recombination reaction
  Linearized carrier	2 μL
  Target gene segment	1 μL
  2 × ClonExpress Mix	5 μL
  ddH2O	2 μL

  Use a pipette to gently suck and mix (do not shake and mix), and briefly centrifuge to collect the reaction liquid to the bottom of the tube. 50℃, 15 min; Reduce to 4 ° C or cool immediately on ice
  
• Transformation of recombinant products
  Thawing chemoreceptor cells for cloning on ice
  Add 10 μl recombinant product into 100 μL receptive cells, gently mix the tube wall (do not shake and mix), and leave it on ice for 30 min.
  After the 42℃ water bath is heated for 45 seconds, it is immediately placed on ice to cool for 3 min.
  Add 900 μL LB liquid medium (without antibiotics) and shake the bacteria at 37℃ for 1 h (rotational speed 200 rpm).
  Preheat the corresponding resistant LB solid medium plate in a 37℃ incubator.
  Centrifuge at 5000 rpm (2,500 × g) for 5 min and discard 900 μL supernatant. The bacteria sediment was suspended with the remaining medium and lightly coated with a sterile coating stick on a plate containing Amp resistance.
  Culture inversely in an incubator at 37℃ for 12 h.
  

Result

Left：plactate1, placate2 Plasmid skeleton amplification size are 3150bp and 3800bp
Right：plactate1-sfGFP, plactate2-sfGFP amplification size are both 750bp