4 Notes
1. We use Quantifoil
® gold 200 mesh EM grids with 2 μm holes
and 1 μm spacing (R2/1). Smaller holes sizes can be used if
higher magnifications than described in this chapter are used
for imaging. We prefer Quantifoil over C-flats as the carbon
film is stronger.
2. We use Dumont, N5-PO, Dumoxel
® . Alternatively, anticapillary tweezers can be used that prevent the uptake of liquid
and thus might enable easier release of the grid.
3. Any grid box similar to the ones from Electron Microscopy
Sciences (EMS), cat #71155 can be used.
4. Mica can be purchased from any supplier of EM consumables,
e.g. EMS, cat # 71855-05.
5. Thickness and grade of carbon rods depend on evaporator
type. Check manufacturer instructions.
6. Prepare medium under sterile conditions in biosafety cabinet.
7. SK-MEL-2 cells were used for work described in this chapter.
However, protocols can be adapted for the use of other cell
lines.
8. We used 10 nm BSA Gold Tracer (EMS, cat # 25486). It is
important to have the gold colloidal conjugated to a protein or
PEG coating. Uncoated gold colloidals aggregate when added
to solutions containing salt.
9. We use Gold Seal Specialty Papers PTFE Sheets from Amazon.
10. We use a Vitrobot Mark IV (Thermo Fisher Scientific, previously FEI). Alternatively, manual plunge freezers or other
automatic devices such as the Leica GP2 or the Gatan Cryoplunge 3 can be used.
11. Small holes are drilled into the lid and top of conical tube to
allow liquid nitrogen gas to escape and to attach string. The
purpose of the string is to retrieve the conical tube from the
liquid nitrogen sample storage dewar. Small metal screws or
bolts can be added to the conical tube to ensure the tube is
submerged in the liquid nitrogen.
12. Cryo-EM grid boxes can be purchased from standard suppliers
of EM consumables (e.g. EMS, cat #71166-40). To open the
grid boxes, use the gripper tool (EMS, cat #71166-SP).
13. It is important to have fast access to data and sufficient RAM to
hold a tomogram in memory (32 or 64 GB are good, 128 GB
is excellent). GPUs enhance the speed of tomogram
reconstructions.
18
Daniel Serwas and Karen M. Davies
1. We use Quantifoil
® gold 200 mesh EM grids with 2 μm holes
and 1 μm spacing (R2/1). Smaller holes sizes can be used if
higher magnifications than described in this chapter are used
for imaging. We prefer Quantifoil over C-flats as the carbon
film is stronger.
2. We use Dumont, N5-PO, Dumoxel
® . Alternatively, anticapillary tweezers can be used that prevent the uptake of liquid
and thus might enable easier release of the grid.
3. Any grid box similar to the ones from Electron Microscopy
Sciences (EMS), cat #71155 can be used.
4. Mica can be purchased from any supplier of EM consumables,
e.g. EMS, cat # 71855-05.
5. Thickness and grade of carbon rods depend on evaporator
type. Check manufacturer instructions.
6. Prepare medium under sterile conditions in biosafety cabinet.
7. SK-MEL-2 cells were used for work described in this chapter.
However, protocols can be adapted for the use of other cell
lines.
8. We used 10 nm BSA Gold Tracer (EMS, cat # 25486). It is
important to have the gold colloidal conjugated to a protein or
PEG coating. Uncoated gold colloidals aggregate when added
to solutions containing salt.
9. We use Gold Seal Specialty Papers PTFE Sheets from Amazon.
10. We use a Vitrobot Mark IV (Thermo Fisher Scientific, previously FEI). Alternatively, manual plunge freezers or other
automatic devices such as the Leica GP2 or the Gatan Cryoplunge 3 can be used.
11. Small holes are drilled into the lid and top of conical tube to
allow liquid nitrogen gas to escape and to attach string. The
purpose of the string is to retrieve the conical tube from the
liquid nitrogen sample storage dewar. Small metal screws or
bolts can be added to the conical tube to ensure the tube is
submerged in the liquid nitrogen.
12. Cryo-EM grid boxes can be purchased from standard suppliers
of EM consumables (e.g. EMS, cat #71166-40). To open the
grid boxes, use the gripper tool (EMS, cat #71166-SP).
13. It is important to have fast access to data and sufficient RAM to
hold a tomogram in memory (32 or 64 GB are good, 128 GB
is excellent). GPUs enhance the speed of tomogram
reconstructions.
18
Daniel Serwas and Karen M. Davies