[3dem] Problems blotting the liquid ethane

[Robert Marshall]

Eduardo,
To be sure all the ethane has evacuated, be sure to pre-pump the  
sample for at least 90 seconds.
Bob

Dept. of Neurobiology
Stanford University
Stanford, CA  94305

On Feb 22, 2008, at 11:23 AM, Bill Tivol wrote:

>
> 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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