[3dem] C-flat grids cryo-EM with manual blotting

[Puey Ounjai]

Hi Jean,
There are several things you can try! Some of them work for me!

1) float a thin layer of C on the C-flat and blot normally
2) vary the drain time (waiting time between blot and plunge)
3) blot from both side
4) use 2-3 layers of dry blotting paper instead of one
Hope that helps,
Puey


On Wed, Dec 10, 2008 at 3:29 AM, Jean Watermeyer
<jeanwatermeyer at gmail.com>wrote:

> Hi all
>
> Is there anyone on this list who has been doing cryo-EM with C-flat grids,
> using manual blotting, and glow-discharging?
>
> We have been trying to optimise our blotting for the thinnest possible ice
> and have found several problems:
>
> - if the blotting is too rough or the plunging too deep, all of the carbon
> comes off the grid
> - if we glow-discharge for 10s at 20 mA, blotting for ~8 s at 50% humidity,
> we get ice that is much too thick
> - without glow-discharging, we get too steep a gradient of ice thickness
>
> We'd really appreciate hearing from anyone who has managed to get very thin
> ice with these grids using manual blotting.
>
> Sincerely
> Jean Watermeyer
>
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