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Freezing apoferritin with the Vitrobot

1. Fill the LN dewar. We need around 4L.

2. Assemble the humidifier on vitrobot machine. Remember to put the correct direction facing towards you (labelled on humidifier). Fill the humidifier with 60ml of distilled water. Do a little de-filling to create the vacuum inside.

3. Turn on vitrobot power. Set temperature to 4°C, humidity 100%. Put blotting paper on the ring.

Tips: We do 1,2 first because humidity building and temperature cooling might take a while.

4. Now we have a chance to dryrun the machine. The purpose is to see if every step runs correctly.

Set blot time 4s, blot force 1, blot total 1, drain time 0. On the miscellaneous panel, check options best fitting your habit. Here I will assume we checked ‘use footpedal’, ‘humidifier off’, ‘skip grid transfer’, ‘autoraise ethanelift’.

Setting should look like this.

Close the hood door. Then press pedal to start the dryrun process. Make sure the pausing time at each step is correct and the blotting paper holders are aligned at the center. You should familiarize yourself what will happen at each step after you press the pedal.

5. Take out frozen apoferritin samples, thawing in ice bucket.

6. Pour liquid nitrogen into coolant container. Do not pour into the center ethane container. Refill several times until the container is completely cooled down. Put desired number of grid boxes into the coolant container. See image.

7. Open the ethane gas tank, and adjust the output pressure to have a weak but steady flow of ethane. Then close the tank for now.

8. Carefully put the glass tip of ethane gas tube inside the ethane container and touch the bottom.

9. Open the tank now and wait for liquid ethane to build up. During this process, you will hear a small noise. That’s the ethane gas coming out. With the liquid surface growing up, the noise will then be bubbles breaking at the surface.

10. After the surface reach near the top of the ethane container, close the gas tank, put glass tip back, remove the spider.

Tips: In some practice, people will cool down the liquid ethane further to have a little solid ethane at the bottom. In this way, liquid ethane will stay at melting point stably.

If you overly cooled down the ethane and too much solid ethane appear, simply put the spider upside down on the ethane container. It will warm up soon.

11. Now we do plasma cleaning for grids. Turn on the power of plasma cleaner (solarus 950). Turn on argon gas tank (25% argon 75% oxygen). The output pressure should be between 20-25 psi. Select recipe ‘test clean’. Press ‘Start’.

12. During the self-clean, put new grids ( for apoferritin, we use xxxx) on glass slides wrapped with parafilm (new products like metal grid holder function the same here). The glass slides should be put inside a petri dish.

13. When ‘test clean’ finish, press ‘stopxxxnotsure’ to release the vacuum. Select recipe ‘John’s recipe’, put the petri dish inside without lid. Press ‘Start’.

14. After the program finish, press ‘stop’. Put the petri dish lid on, they are ready for vitrobot. Turn off the plasma cleaner, and close the argon gas tank.

15. Now we return to vitrobot workstation with our freshly cleaned grids and thawed samples.

16. Refill LN into coolant container if necessary. Put the coolant container on vitrobot.

17. Carefully use the tweezer to grab a grid. Lower the black rubber ring to lock the tweezer. See image below.

18. Assemble the tweezer on vitrobot. You will hear a sound of click. That means the tweezer is at correct position. Have your micropipette ready. We can start the vitrification now.

19. Press the pedal. The tweezer will be put inside the hood. The coolant container will be raised to cover the bottom hole of vitrobot. See image.

20. Press the pedal. The tweezer will be lowered a little bit. Open the side sample application hole, take 3.5 uL apoferritin (4mg/ml) using micropipette. Insert the micropipette tip into the hood through the side hole. While observing the grids by eyes, slowly apply all samples on the grid. A good tip is to rotate your micropipette so that samples are evenly applied on grid. After applying, take micropipette out, close the side hole. See image.

21. Press the pedal. The tweezer will be raised. Blot paper will be put on the grids. After 4 seconds, blot papers are released. The bottom hole will open, and the tweezer will be inserted into ethane abruptly. The tweezer together with coolant container will be lowered slowly onto table.

22. Fill the coolant container so that the anti-contamination ring rise to proper height. See image.

23. Carefully remove tweezer. Avoid hitting your grid on any surface. Then quickly move grids from ethane into liquid nitrogen and carefully put the grid inside grid box. If this is tricky for you, uncheck the ‘skip grid transfer’ option, and move tweezer together with the coolant container out of vitrobot to process.

24. A grid is successfully vitrified now. Simply repeat this process (16-23) for more grids.

25. After you finish, close the grid boxes lids. Transfer the grid boxes to your desired container (centrifuge tube, Mitegen grid storage puck).

26. Now is cleaning. Coolant container should be put inside a minus pressure hood. Put the tip protector on tweezers and put them back in tool box. Open the vitrobot hood, remove blotting paper, leave the blot paper lock ring inside. Turn off vitrobot. Remove humidifier, put it upside down to drain water.

27. Remove any trash, and clean the table.


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