[3dem] Contamination problem on cryo grids

Anselm Kusser kusser at lmb.uni-muenchen.de
Fri Sep 19 04:41:33 PDT 2008


We are repeatedly experiencing problems with significant amount of contamination on our grids (follow the links to have a look at some example images).

http://www.cip.ifi.lmu.de/~kusser/CCD_search_01.jpg
http://www.cip.ifi.lmu.de/~kusser/CCD_search_02.jpg
http://www.cip.ifi.lmu.de/~kusser/CCD_hole_01.jpg
http://www.cip.ifi.lmu.de/~kusser/CCD_hole_02.jpg


I will try to describe the conditions we use:

buffer composition:
10 mM Hepes ph 7.8
40 mM Ammonium Sulfate
0.5 mM Mg Cl2
10 uM Zn Cl2
10 mM DTT

protein concentration:
~100µg/ml

grids:
Quantifoil precoated, Typ R3/3, Cu 300 mesh, with 2nm Carbon on top

glow discharging:
wash with Chloroform, then glow discharge 30s @ 2.2x10e-1 torr using a plasma cleaner

freezing (FEI Vitrobot, 4°C, 95% hum.):
apply 3,5µl of sample to grid, incubate 45s, blot 10s, plunge into liquid Ethane, transfer in N2 gas phase to grid box (also in gas phase) - but contaminations also occur when working in liquid N2 phase while for other poeple/samples gas phase transfer works fine (that was our first suspicion)

transfer to microscope:
transfer one single grid on a Gatan Cryo-Workstation to a Gatan Cryo-Holder, then insert into side entry stage microscope (FEI 120 kV Spirit)

Much oblidged for any suggestions.

Anselm


------------------------------------------------------------------------
Anselm Kusser
Gene Center
Department of Chemistry and Biochemistry
Ludwigs-Maximilians-University of Munich
Feodor-Lynen-Str. 25
81377 München
kusser at lmb.uni-muenchen.de









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