[3dem] Contamination problem on cryo grids

[Willem Tichelaar]

Dear Anselm,

With thin carbon film on home-made, conventional holey film, I obtained 
significantly less of those thick ice blobs, when I used another 
glow-discharge device at the EMBL.

Good luck,
Willem Tichelaar.


Anselm Kusser wrote:
> 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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>   

-- 
Willem Tichelaar, Ph.D.
European Molecular Biology Laboratory
Meyerhofstr. 1
69117 Heidelberg, Germany

phone: +49 (0)6221 387 567; fax: +49 (0)6221 387 519