[3dem] Contamination problem on cryo grids

[Shi, Dan (NIH/NCI) [C]]

Anselm,
I agree with Bob. The contamination most likely comes from a 'dirty' grid. You may be able to clean out the dirty spots by using acetone, chloroform and plasma treating, and check the quantifoil grids before and after clean procedures to find out if any one of them works. Anther possible sources might be one of the chemical agents you used was 'sticky' to some uneven discharged 'pockets' to form the 'splotchy ice'. You can try to use ethanol environment for glow-discharging the grids to make the carbon surface strong hydrophilic. Good luck,
Regards,

Dan Shi, PhD
LCB, National Cancer Institute, NIH

-----Original Message-----
From: rg2502 at columbia.edu [mailto:rg2502 at columbia.edu] 
Sent: Friday, September 19, 2008 9:36 AM
To: kusser at lmb.uni-muenchen.de
Cc: 3dem at ucsd.edu
Subject: Re: [3dem] Contamination problem on cryo grids

Hi Anselm,
What you are seeing is not contamination, it is what we term "splotchy  
ice" which occurs if there are contaminants on the carbon causing the  
carbon to be hydrophobis not hydrophyllic.  We routinely treat our  
Quantifoil grids with chloroform environment to eliminate the  
offending contaminant.  I see that you have done this so it may me a  
water soluble contaminant so try washing it with water or a detergent.  
  Unfortunately this is a problem that is well known to Quantifoil  
grid users but is easily overcome.
If this is not a solution you may be getting oil from you glow  
discharge device or making the thin 20 nm carbon.  Is the bell jar of  
both devices clean?  Has anyone else experienced the same problem  
recently (problem with equipment).  In my experience when this type of  
problem happens there is something bad about the carbon.  Good luck.
Bob

Quoting Anselm Kusser <kusser at lmb.uni-muenchen.de>:

> 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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