[3dem] Problems blotting the liquid ethane
Robert Marshall
rmarshal at bluebottle.com
Fri Feb 22 12:30:16 PST 2008
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
>
> _______________________________________________
> 3dem mailing list
> 3dem at ncmir.ucsd.edu
> https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
-------------- next part --------------
An HTML attachment was scrubbed...
URL: https://mail.ncmir.ucsd.edu/mailman/private/3dem/attachments/20080222/9a452224/attachment.html
More information about the 3dem
mailing list