CSB CryoEM Facility TF20 Operating Instructions SEC
05/02/16
13.
Verify that the clip ring is securely held in place with the cold black rod (clipring should not
move). If clipring is loose, cool down the knob end of the clipring tool and gently press on the
clip ring.
14.
Close the shield on the cryo holder and remove the tools from the transfer station.
15.
Continue adding LN2 as needed to the cryo transfer station but do not overflow.
16.
Tighten the grid box lid and remove from transfer station.
17.
Carefully remove the cryo holder from the transfer station and insert it into the microscope.
18.
The guide pin on the cryo holder will be at the 12 o’clock position and must be quickly rotated
clockwise to the 3 o’clock position so that the airlock is triggered and the pre
pump begins. When the position of the holder is correct, the holder will slide into the airlock
about 1 inch further.
19.
Select the ST Cryo holder option (bottom of the Tecnai User Interface).
20.
Wait for the airlock pumping cycle to finish (6090s) as indicated by the timer on the UI.
21.
When the holder is upright, reset the stage tilt to 0° by going to the Search tab, flapout window,
Control tab, Alpha toggle and click Set Alpha (this will return the stage to 0°).
22.
Gently allow the vacuum to pull the holder into position.
23.
Refill the dewar on the cryo holder. Use the debubbler (rubber stopper with tubing) to remove
the any excess LN2 from the neck of the cryoholder.
24.
Remove any remaining LN2 from the transfer station and allow the tools used to warm up and
dry off.
25.
Wait ~1015 minutes before opening the column valves. Because there is a lot of movement
happening during insertion, the grid will drift a lot until it settles down. Ideally the column
vacuum (IGP1) will return to 6 before viewing the sample.
