[3dem] specimen preparation

Bob Grassucci rg2502 at columbia.edu
Wed Nov 6 06:28:10 PST 2013


Hi Kimberly,
Which Version of the Vitrobot do you have?  With the Mark IV model they 
changed the mechanism that adjusts the blot force.  They now adjust the 
distance that the blotting pads from each other and unfortunately this 
can change over time.  You want to blot with a more positive force and 
if that does not work blot longer.  This should help with sample 1.

For sample 2 to get an increase in sample concentration increase the 
wait time.  We use 30 seconds for most of our ribosomes .  We use thin 
carbon over the holes.  I hope these suggestions helpRegards,
Bob

On 11/6/2013 8:34 AM, Kimberley Cheng wrote:
> Dear all,
>
> I have difficulties preparing cryoEM specimens of my samples, and I 
> would appreciate any help I can get. Both samples contain lipid 
> nanoparticles, and I use a Vitrobot to prepare the frozen-hydrated 
> specimens. The problems are the following
>
> Sample 1: Majority of the grid has no ice at all. If I am lucky, a 
> small region of the grid (1/4 of a grid square) is covered with ice 
> that is very thick. What can be the problem? Any advice on how to 
> improve the procedure?
>
> Sample 2: Ice thickness and particle distribution is good. The 
> concentration is, however, very low. Any suggestions on how to 
> increase the on-grid particle concentration? I have already tried 
> preparing the sample on continuous carbon instead of perforated carbon 
> film.
>
> I am very interested in hearing your opinions in this.
>
> Thanks in advance,
> Kimberley Cheng
>
>
>
>
>
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