[3dem] specimen preparation

[Bertin Aurelie]

Dear Kimberly,
Maybe you have an issue with the glow discharge conditions. The inhomogeneity (point 1) might result from that.
For point 2, you can try to incubate your sample in a humid athmosphere (wet closed box) for several minutes up to an hour before freezing it.
Best
Aurelie

-----Message d'origine-----
De : 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] De la part de Bob Grassucci
Envoyé : mercredi 6 novembre 2013 15:28
À : 3dem at ncmir.ucsd.edu
Objet : Re: [3dem] specimen preparation

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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