[3dem] Problems blotting the liquid ethane

[Bill Tivol]

On Feb 22, 2008, at 9:50 AM, Eduardo Sanz-Garcia wrote:

> I am having real problems to blot successfully the liquid ethane  
> from my samples after freezing.
> I am using a vitrobot for freezing. This is very neat because makes  
> everything automatic except the blotting of the liquid ethane.
> I know that I should see a drop of ethane in the filter paper, but  
> this step has been very irregular for me (very few times I see  
> filter paper wet).
>
> Do you have any suggestion for me?
>
> Does the speed of the process affect (remove the sample from the  
> ethane, blot and put in the cryo box) the rate of success?
> Could be that the temperature around the filter paper is too low  
> (nitrogen temperature) and that the ethane solidifies before  
> touching the filter paper?
> For how long should I precool the filter paper?


Dear Eduardo,
	I don't blot the liquid ethane at all. When I remove the tweezer  
from the ethane and dip it into LN2, the ethane freezes.  The ethane  
that remains on the grid protects against ice crystals landing on the  
grid during the transfer to the grid box, and the ethane is gone by  
the time the grid is loaded into the scope.  Furthermore, ethane is  
non-polar, so it would not be expected to wick into the filter paper,  
which is cellulose and, therefore, polar.
Yours,
Bill Tivol, PhD
EM Scientist
Electron Cryo-Microscopy Facility
Broad Center, Mail Code 114-96
California Institute of Technology
Pasadena CA 91125
(626) 395-8833
tivol at caltech.edu

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