Centrifugation

• • 250ml bacterial secondary culture and 4-6 autoclaved Oakridge tubes are placed inside the laminar hood.
• • 250 ml secondary culture is poured into 4-6 Oakridge tubes equally.
• • 4-6 Oakridge tubes are centrifuged at 8000 rpm at 4℃ for 30 min to pellet down the bacteria.

Filtration:

• • Membrane filtration is done for the supernatant with 0.22 micron filter
• • After that supernatant is collected into an autoclaved conical flask for ultracentrifugation.

Weighing:

• • Polycarbonate tubes: not autoclavable, have to wash only with distilled water.
• • Supernatants have to pour up to the mark ( aprox 90%of the tub, remaining 10% to grip the tube and to make sure that supernatant does not fall. Less than 90% will result in breaking of the tubes in ultracentrifuge)
• • First take a beaker in the weighing machine and tare the weight.
• • Next, put the beckman coulter tube’s cap into the weighing machine on the side.
• • Supernatant is filled into the polycarbonate tubes up to the mark and then put into the beckman coulter tubes.
• • Beckman Coulter tubes with polycarbonate tubes are placed into the center of the machine while a cap is also present into the machine and weight is taken.
• • This procedure is repeated for all tubes and adjusted until the weight of all tubes become equal up to two decimal points.
• • After that the tubes are weighed again for three times to recheck whether the weight is the same.

Ultracentrifugation:

• • Ultracentrifuge: 25000rpm, 4℃, 4 hours
• • After 4 hours, let the rpm speed keep reducing, there would be a sound.
• • Then, touch the vacuum button in screen, the vacuum would increase >1000 um
• • Once it's done there would be another confirmation sound played.
• • Next, gently slide open the lid and take out the rotor with the cassettes attached.

PBS wash:

• • Discard the supernatant from each of the 6 tubes.
• • Add 0.5-1 ml of 1X PBS buffer to one of the tubes and pipette up and down in order to dissolve the OMVs into the buffer.
• • Pipette out the dissolved OMV + buffer and pour into the next tube. Try shooting the PBS buffer in the place where OMVs have aggregated.
• • Then collect the OMV-dissolved PBS buffer into the MCT tubes (1.5-2 ml)
• • Again, rinse with 0.5-1 ml of 1X PBS buffer and collect in another MCT.
• • Store the MCTs at -20℃.

OMV Purification:

• 1. Thaw the stored isolated OMV in MCTs on ice.
• 2. Polycarbonate tubes: not autoclavable, have to wash only with distilled water and keep under UV for 5mins.

DSF Isolation: (8)

• 1. Inoculate the strain in primary culture with antibiotics
• 2. Grow to saturation at 28C
• 3. Inoculate for secondary culture in media without antibiotics
• 4. Grow to saturation at 28C
• 5. Pellet the cells at 8000rpm for 10minutes at room temperature
• 6. Collect the supernatant
• 7. Add water saturated ethyl acetate to the supernatant in 5:2 ratio
• 8. Stir with magnetic beads for atleast 45-60 minutes
• 9. Centrifuge at 8000rpm for 10 minutes at room temperature
• 10. Take the upper layer and dry with speed vacuum
• 11. Dissolve the extracted DSF in methanol(i.e, 250ul methanol for 250ml culture)
• • Preparation of water saturated ethyl acetate
• 1. Take 100ml of ethyl acetate
• 2. Add 50ml of autoclaved MilliQ water to it
• 3. Stir for 10-15minutes with magnetic beads
• 4. Keep in a reagent bottle(wrapped with aluminium foil to prevent light exposure ) at room temperature

• • Give 3mL Xanthomonas primary culture in 2.5% LB, incubate for 16-20 hours at 28°C and 250rpm.
• • Give 30mL secondary culture in 2.5% LB with 300uL inoculum, incubate at 28°C for 3.5hours at 250rpm.
• • Transfer the culture to an Oakridge tube and centrifuge at 6000rpm for 5minutes at 4°C. Discard supernatant and resuspend pellet with 30mL dH2O.
• • Centrifuge at 6000rpm for 8minutes at 4°C. Discard supernatant and resuspend pellet in 15mL dH2O.
• • Centrifuge at 6000rpm for 8minutes at 4°C. Discard supernatant and resuspend pellet in 5mL dH2O.
• •Centrifuge at 6000rpm for 8minutes at 4°C. Discard supernatant and resuspend pellet in 100uL dH2O. Entire pellet will not dissolve, dissolve as much as possible and transfer 100uL of cell culture to a 1.5mL MCT placed in ice bucket.
• • Transfer 100ng of plasmid to the cell culture and keep for 15minutes. Meanwhile, cool the electrocuvette at 4°C.
• • After 15minutes, transfer 100uL of the mixture to the electrocuvette and again keep in 4°C for 15minutes.
• • Electrocute the mixture inside a cuvette using a pulse voltage.
• • Quickly add 1mL of LB inside the electrocuvette and mix well via pipetting.
• • Transfer 1mL of the culture to a fresh 1.5mL autoclaved MCT. Incubate it for 3.5hours at 28°C and 250rpm
• •Centrifuge the solution at 6000rpm for 5minutes. Discard supernatant and resuspend pellet in 100uL LB.
• • Spread the 100uL culture on an antibiotic LB agar plate and incubate for 1-2 days at 28°C.