[3dem] Breaking of C-flat carbon and vitrification

[Terje Dokland]

We also found that the C-flat film is very fragile. We have been  
unable to use the 200 mesh grids, even with the double-thick carbon,  
although we do use the 400 mesh C-flat grids routinely and they are  
generally ok.

We also found that it is important not to over-glow discharge the  
grids to get good sample thickness. The default setting on our glow  
discharger was 100mA for 30 sec, but I reduced it to 25mA for 10 sec  
for grids that we use for cryo-EM. (For negative staining, we use  
50mA for 20 sec) This is for using with one-sided (manual) blotting  
and not with the Vitrobot, though, so your optimal settings may be  
different.


On Aug 6, 2010, at 11:38 AM, M.Karuppasamy at lumc.nl wrote:

> Dear All,
>
>
>
> Could you help us on how to get optimal & reproducible (thickness)  
> samples for SP cryo-EM?
>
>
>
> We use a FEI vitrobot mark IV, typically with the following  
> blotting parameters: C-flat support, 3.5 ul sample per (30sec glow  
> discharged) grid, 3 sec blotting time, blotting force 3, 100%  
> humidity, RT.
>
>
>
> Unfortunately, we frequently notice the breaking of C-flat carbon  
> support during vitrification. Example images are attached.
>
> Only very few meshes are left after vitrification. In those meshes,  
> sample is only found in the middle of the mesh and generally it is  
> still too thick (150->200nm). We played around with blotting  
> parameters but didn’t find optimal ones yet for our samples.
>
>
>
> Thanks & regards
>
>
>
> Manikandan K.
>
>
>
>
>
> <overview1.jpg><overview2.jpg><ATT00001..txt>

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