Vitrobot SOP

Preparation of the Vitrobot

 * 1) Turn on the Vitrobot, wait the booting sequence to finish.
 * 2) If desired, mount the humidifer and fill it through the tubing at its bottom with 15-20 mL of pure water. This is enough for a few hours of operation. It is recommended to not over fill it.
 * 3) Setup the parameters on the touch screen.
 * Typical settings: 5°C, 100% humidity, 1-3s of blotting, 10s waiting (time between adding the drop to grid and the blotting action).

Preparation of the cryogen
Warnings: 

Although liquid nitrogen is unlikely to cause serious frostbite owing to Leidenfrost effect, liquid ethane's boiling point is -89°C and liquid propane's boilding point is -42°C. It is possible for them to cause serious injuries to human body, especially to the eyes. Safety goggles should be worn when working with these cryogens.

Ethane and Propane are highly flammable in gaseous state. Their evaporation at cryogenic temperature is negligible.


 * 1) Assemble the Styrofoam container, place the aluminium spider.
 * 2) Pour some liquid nitrogen into both the copper cup and the Styrofoam container. Then keep filling the Styrofoam container but not the copper cup. In the end the copper cup should become empty and the Styrofoam container is filled with liquid nitrogen.
 * 3) Open the main valve of the ethane tank and the small value on the regulator. Slowly turn the regulator knob to let the ethane start flowing. One can check the flow by directing the nozzle towards skin. Once ethane starts to flow, push the nozzle against the bottom of the copper cup and slowly fill the cup with liquid ethane. A strong flash light could be very helpful to see the ethane level.
 * 4) When the ethane reaches the bottom of the spider, turn the regulator knob to reduce the flow and lift the nozzle. Then turn the regulator completely off. If the flow is completely off before lifting the nozzle there is a chance that the ethane in the tubing will be cooled generating low pressure and suck the liquid ethane into the tubing.
 * 5) Put a 5 cent coin on top of the spider to melt the frozen ethane between the spider and the copper cup. Remove the spider with a large tweezer. Do not put the cryogen container on the Vitrobot yet (need to leave room for mounting the tweezer).

Plunge freezing
'''Keep the working area orderly to minimize chance of accidents. The $1000-tweezer tip is extremely delicate.'''
 * 1) Some checks:
 * 2) Check the settings on the touch screen.
 * 3) Check that the ethane is not completely frozen. If it does, warm it with a clean tweezer or a metal rod.
 * 4) Put a cryo grid box in the liquid nitrogen
 * 5) Check that the aluminum spider has been removed.
 * 6) New users can uncheck "Skip grid transfer". This will allow the Vitrobot to lift the grid from the ethane cup and move it over the liquid nitrogen.
 * 7) Pick a glow-discharged grid with the tweezer with dovetail groove. Mount the tweezer on the end of the rod on Vitrobot. Remember to let the holey carbon film side face the side you will load protein from.
 * 8) Lift the tweezer from the touch screen.
 * 9) Check that the aluminum spider has been removed. Put the cryogen container on the holder.
 * 10) When both the tweezer and the container are at right position, load a drop of protein to the grid from the small opening on the side of the vitrobot. One might want to check "humidifier off during blotting".
 * 11) Click the touch screen to let Vitrobot proceed through the plunge freezing.
 * 12) As stated above, one can let the Vitrobot to lift the grid from the copper cup at the end. The grid will stop in the cold nitrogen gas layer.
 * 13) Carefully remove the tweezer and store the grid in the cryo grid box.

Cleaning up

 * 1) Check both valves on the ethane tank and regulator are closed. The knob is turned to the maximum height (least opening).
 * 2) Remove humidifier and empty the water. Leave it to dry.
 * 3) Move the cryogen container to a hood. Pour out the liquid nitrogen and leave the ethane to warm up and evaporate in the hood. Never let the ethane to evaporate in a non-ventilated room
 * 4) Turn the Vitrobot off (from touch screen and then switch at back).
 * 5) Clean up the work area. Safely store the tweezer.