Experiments


Experiments


Protocols


PCR :

  1. Extract 2 ul of sample/DNA, mix it with 2ul of reverse primer, 2ul of forward primer, 25ul of Phanta Flash Master Mix and 19ul of dd h3O as buffer in a clean, sterile tube.
  2. Place the tube containing the reaction mixture in the thermal cycler and start the program. The thermal cycler will cycle through denaturation, annealing, and extension steps multiple times. The number of cycles depends on the starting amount of DNA and the desired level of amplification.
  3. After the PCR reaction is complete，using gel electrophoresis to visualize the amplified DNA fragments and determine their size.


Gel preparation:

  1. Put 0.4g agarose into a conical flask and mix it with 40mL TAE solution. Then, microwave the mixture for 1 minute.
  2. Add 4ul nucleic acid dye into the mixture and shake the mixture gently.
  3. Finally, decant the gel into adhesive board and insert a gel comb with appropriate number of holes.
  4. Wait for around 10 minutes.


Agarose Gel Electrophoresis :

  1. Add 1/10 of sample loading buffer into the sample and put the gel into the electrophoresis tank. Then, add sufficient buffer which could cover the gel.
  2. Then, add the sample into the gel with moderate amount of DNA marker. Wait 30 minutes to let the DNA run out.


Cutting Gel:

  1. Visualize the gel under UV light in a gel documentation system . The DNA bands will appear as fluorescent spots.
  2. Then put the gel in a handheld UV lamp. Put on appropriate protective gear such as gloves and safety glasses to protect against UV radiation.
  3. Using a clean scalpel or a gel cutting tool, carefully cut out the desired band of DNA. Try to make clean cuts to minimize contamination and damage to the DNA. And put the gel with DNA into centrifuge tubes separately.


Gel extraction:

  1. Add three times the volume of the gel of PE solution to the gel cut off seperately and melt the gel in room temperature for around 10 minutes.
  2. Transfer the solution obtained into adsorption column CA5 and place the columns for 2 minutes in room temperature. Then centrifuge the solutions at a speed of 12000rpm for 60 seconds. Then pour out the waste in the collection tubes.
  3. Add 600 ul of Buffer-PW into adsorption column CA5 and centrifuge the solutions at a speed of 12000rpm for 60 seconds. Then pour out the waste in the collection tubes and put the adsorption column CA5 into the collection tubes.
  4. Repeated step 3. Put the adsorption column CA5 into the collection tubes to centrifuge the solutions at a speed of 12000rpm for 60 secongs until no Buffer-PW remains.
  5. Place the adsorption column CA5 in room temperature for few mintues until the adsorption column CA5 are completely dry.
  6. Put the adsorption column CA5 into clean centrifuge tubes and add 30 ul Elution buffer TB and place the solution in room temperature for 2 minutes. Then centrifuge the solution for 2 minutes at a speed of 12000 rpm.
  7. Examine the purity of the DNA extracted.


Gibson assembly

  1. Replicate the DNA fragment that required to be assembled by PCR.
  2. Cut the 5’ end of the DNA fragment.
  3. Stack the single-stranded DNA fragments together and assemble them by base pairing.
  4. Fixing the unpaired bases by DNA polymerase and DNA ligase.
  5. Transfer the assembled DNA fragment into the host cell.


Transformation

  1. Mix the chemically competent cell with plasmid DNA.
  2. Incubate the cells with DNA on ice for 5‒30 minutes.
  3. Perform heat shock at 37‒42°C for 25‒45 seconds as appropriate for the bacterial strain and DNA used.
  4. Return the heat-shocked cells to the ice for more than two minutes.


Bacteria culturing

  1. Prepare the medium with appropriate antibiotic.
  2. Prepare the cell suspension that required to be cultured or screened.
  3. Spread the cell suspension with a cell spreader.
  4. Incubate the plate in an incubator at an appropriate temperature.


Bacterial colony PCR

  1. Mix 2ul of reverse primer, 2ul of forward primer, 25ul of Phanta Flash Master Mix and 21ul of dd h3O as buffer in a clean, sterile tube.
  2. Choose few single colonies and mark them on the plates.
  3. Add the colonies to the PCR system.
  4. Place the tube containing the reaction mixture in the thermal cycler and start the program. The thermal cycler will cycle through denaturation, annealing, and extension steps multiple times. The number of cycles depends on the starting amount of DNA and the desired level of amplification.
  5. After the PCR reaction is complete，using gel electrophoresis to visualize the amplified DNA fragments and determine their size.


Liquid culture of bacteria

  1. Prepare liquid LB medium with appropriate antibiotics.
  2. Choose single colony and add to the the LB medium with the tip of pipette.
  3. Blend the colony with LB medium by pipette tip.
  4. Cover the culturing container with bacteria-free aluminum foil or a non-hermetic cover.
  5. Culture the bacteria in a shaking incubator at an appropriate temperature.


Plasmid extraction

  1. Cultivate bacterial samples overnight in growth medium with antibiotic selective pressure.
  2. The bacterial culture is centrifuged and the supernatant is discarded.
  3. A buffer is added to the tube containing bacterial pellet in order to resuspend the pellet.
  4. Add the lysis buffer and gently invert the tube multiple times to fully lyse the cells.
  5. Neutralization buffer (typically potassium acetate solution) is added and gently inverted to neutralize the alkaline conditions and lower the pH.
  6. The mixture is then centrifuged, where genomic DNA and precipitated proteins form a pellet and the plasmid DNA remains in the supernatant.
  7. Add 100% ethanol to the supernatant in order to precipitate plasmid DNA. Once precipitated, the precipitate containing the plasmid DNA is rinsed in 70% ethanol to remove any remaining salts and impurities.
  8. Leave the tube open to remove all the residue alcohol by evaporation.
  9. Resuspend the DNA pellet in a buffer solution containing Tris and EDTA.
  10. Pass washing buffer through the column while the DNA remains bound to the silica.
  11. Repeat the washing step to remove endonucleases, RNA, proteins, dyes, and low-molecular weight impurities.
  12. Elute the plasmid DNA with sterile water or an elution buffer.


Yeast culture

  1. Inoculate yeast into medium under sterile conditions. The inoculation can be performed using tools such as an inoculation ring, an inoculation needle, or a pipet.
  2. Single colonies of yeast recipient bacteria were inoculated on YPD plates and cultured for 2 days at 30 ° C.


Preparation for competent cell of yeast

  1. Single colonies from the plates were picked and inoculated in 10ml YPD liquid medium and shaken overnight at 30°C on a shaker.
  2. After overnight culture, the cells were inoculated into 100ml YPD medium at about 1% inoculum and cultured by shaking to an OD value of about 1.2.
  3. The precipitated bacteria were collected by centrifugation at 5000 rpm for 5min at 4°C and resuspended in 100ml of precooled sterile water.
  4. The precipitated bacteria were collected again by centrifugation at 5000rpm for 10min at 4°C and resuspended in 100ml of precooled sterile water
  5. The cells were washed with 20ml, 1mol/L sorbitol.
  6. The cells were dissolved in 200μl, 1mol/L pre-cooled sorbitol without glycerol and placed at -80°C for several hours until transformation.


Transformation of Yeast

  1. About 100ng DNA (plasmid) was added to the competent cells, and the mixed solution was aspirated by pipettor.
  2. PEG at 600μl was added and mixed at high speed (10S-1 min).
  3. Recovery was performed at 30°C with shaking for 30min.
  4. 70μl DMSO was added to the ultra-clean table (under sterile conditions), and the mixture was gently reversed.
  5. Heat shock in 42°C for 15min.
  6. Leave on ice for 1-2min.
  7. The samples were centrifuged at 14000rpm for 15s and the supernatant was discarded.
  8. Cells were resuspended in 300μl of 1×TE.
  9. 100μl were plated on TPDA plates and incubated at 30°C for 48 to 72h to observe the transformed colonies.


Bacterial PCR for Yeast

  1. Mix 2ul of reverse primer, 2ul of forward primer, 25ul of Phanta Flash Master Mix and 21ul of dd h3O as buffer in a clean, sterile tube.
  2. Choose few single colonies and mark them on the plates.
  3. Add M5 Hiper Super Speed Mix directly amplifies the best companion to the colonies in a tube.
  4. Centrifuge the colonies with M5 Hiper Super Speed Mix transiently and use 1-2ul as PCR template.
  5. Place the tube containing the reaction mixture in the thermal cycler and start the program. The thermal cycler will cycle through denaturation, annealing, and extension steps multiple times. The number of cycles depends on the starting amount of DNA and the desired level of amplification.
  6. After the PCR reaction is complete，using gel electrophoresis to visualize the amplified DNA fragments and determine their size.


Inoculation

  1. Add 90ul bacteria-free liquid (dd h3O or liquid LB medium without antibiotics) into 8 PCR tubes separately.
  2. Add 10ul culture material of bacteria to the first PCR tube and mix it with dd h3O with a pipette.
  3. Obtain 10ul of the cell suspension from the first tube and add it to the second PCR tube. Then mix the liquid with pipette.
  4. Continuing the serial dilution and the concentration of the cell suspension in the last (8th) tube is 10^(-8).
  5. Obtain 1.5ul of the cell suspension from each tube and inoculate on the agar LB plate with appropriate antibiotics.


Coercive evolution

  1. 1. Add 100uM sodium chlorite into 3ml liquid LB medium.
  2. 2. Add ampicillin antibiotics and 50ul previous E.coli bacteria solution.
  3. 3. Add 3ul 1ug/ml Anhydrotetracycline Hydrochloride(aTc) for inducement.
  4. 4. Culture overnight under appropriate conditions.


Culture of Cyanobacteria

  1. Prepare 20ml 1.7g/L BG11 medium with 100mM sodium bicarbonate (by mixing 0.16802g sodium bicarbonate and 0.034g BG11 medium with 20ml water).
  2. Culture 3ul Cyanobacteria sample in 5ml BG11 medium within a centrifuge tube for few days under the light source, such as sunlight or light box.
  3. Examine the OD600 of cyanobacteria every day.


Screening of nitrogen fixing bacteria

  1. Obtain 1g of root nodule or soil sample and add to 20ml dd h3O within a 50ml centrifuge tube. Grind the sample if sample is Obtain 1g of root nodule or soil sample and add to 20ml dd h3O within a 50ml centrifuge tube. Grind the sample if the sample is a root nodule.
  2. Shake the cell suspension and stew for 10 minutes.
  3. Dilute the cell suspension 1000 times.
  4. Spread 40ul 1x, 100x, 1000x cell suspension on the Ashby Mannitol Agar Medium plates with sodium chlorite separately.
  5. Culture overnight at 30°C.


Extraction of plant tissue protein:

  1. Weight 100mg arabidopsis leaf in a 2ml EP tube, crushed after freeze by liquid nitrogen.
  2. Add 250ul RIPA lysis buffer(Thermofisher 89901), 1ul Pierce™ Universal Nuclease(Thermofisher 88700) , 2.5ul Halt™ Protease Inhibitor Cocktail, EDTA-Free(Thermofisher 87785) to the tissue powder, incubated at 4℃ for 1hr.
  3. Centrifuge at 13000rpm for 5min, keep the supernatent.


Western blot detection of GFP and actin protein(internal reference) in plant tissue

  1. Add 30ul extracted protein with 10ul 4X SDS-sampling buffer, heat at 95℃ for 5min.
  2. Loaded 30ul to the 10% Bio Rad precast PAGE gel. Run at 180V for 50min.
  3. Semi-dry transfer at 15V for 30min to a PVDF membrane.
  4. Blot with 1:5000 dilution of primary antibody: Rabbit anti GFP-Tag mAb (Abclonal, AE078) and Actin(Plant Specific) Rabbit mAb (Abclonal, A23959) at room temperature for 2 hours.
  5. Detected by Pierce™ Fast Western Blot Kit, ECL Substrate(Thermofisher, 35050). And record the image with chemi dot instrument.


Detect the firefly luciferase in plant tissue:

  1. Spray the firefly luciferase substract working solution: D-Luciferin, Potassium Salt solution (10 mg/mL in D-PBS) on leaf surface.
  2. Incubate at dark for 10min.
  3. Image with a chemi dot imager or animal live imager.


Seed soaking transformation with TTTT:

  1. Prepare transformation mixture (1X smart-CD(use the stock one as 20X); 10mM MES buffer PH=5.5; 0.5% Glycerol; 5-10ng/ul endotoxin free transformation vector), incubate at 37℃ for 30min, cool down to room temperature.
  2. Clean the seeds with Ethanol/bleach for 30 seconds and wash with distilled water.
  3. Add transformation mixture around 1:1 volume ratio with seeds. Incubate at 25℃ for 24hr, change the transformation mixture and incubate another 24hr.
  4. Plant the germinated seeds into soil.