Wet lab protocols

Tip-Sonicator Protocol

1. Use lipids stocks that were prepared following the Protocols for Preparing Lipid Stocks in the Protocols>Lipids.

2. Calculate the right volume of your lipid stock ( lipids inc chloroform-green lid vial) to transfer to a black lid vial. For accurate lipid concentrations you can measure the empty vial + lid. 

3. Dry the lipids using the Nitrogen stream and put in a desiccator for at least 30 min (depending on the volume)

4. Hydrate the dry lipid film using the correct amount of buffer to reach the desired vesicle concentration. 

5. Vortex the vial for 1 minute. 

6. Fill up a 100 mL beaker with crushed ice. Fill with MQ and this is your ice water, which prevents the tip sonicator from heating up the surroundings.

7. Choose the correct tip! Dirty (for fluorophores), Clean (for no fluorophores), Super Clean (fo single molecule).

8. Prepare the chosen tip-sonicator by cleaning the tip in MQ + 1 minute sonication and then in methanol (100%) + 1 minute sonication. For the MQ and Methanol, use the 10 mL glass vials. Place the glass vials in the ice water so that the level of the MQ, Methanol or your sample is under the level of the ice water.

9. Now you can place the vial with the vortexed vesicles on the clamp and make sure the volume is under the ice water. The tip-sonicator needs to be carefully lowered into the sample, without touching the neck of the vial, especially when you are doing single molecule experiments. Make sure the tip sonicator is positioned in the centre and NOT touching the glass vial anywhere. If the tip touches the glass and the sonicator is turned on, the vial WILL BREAK due to the high frequency vibrations.

10. After meaking sure the setup is ready, turn on the sonicator. Leave for 10 -12 minutes. The cloudy vesicle solution should become clear. 

11. Remove the vial from the setup carefully and transfer the contents into a 1.5 mL eppendorf vial. Centrifuge for 3 minutes at 14.5 RPM. This will cause the TiO2 particles that were released from the tip to the sample, to sediment.

12. Meanwhile, clean the tip AGAIN following step 8. 

13. Remove the supernatant of the centrifuged sample wihtout disturbing the TiO2 particles pellet. 

14. The supernatant is the SUVs solution (<100nm) and now you can process it further as needed.