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

[Matt Swulius]

Terje's response pretty much sums up my experience with c-flat. The 200 mesh
grids simply fell apart no matter how I blotted them. I tried evaporating
additional carbon on to them (as suggested by the manufacturer), but it has
little effect if any. They were very generous though and had a new batch
sent to me free of charge and they gave me some 400 mesh grids to try as
well. As others have noted the carbon support is more durable on the 400
mesh grids, but I was still surprised to see 50% or more of the carbon gone.
I've since stuck with quantifoil and have had no problems getting thin ice.

Matt Swulius
UT Health Science Center - Houston

On Wed, Dec 10, 2008 at 11:46 AM, Puey Ounjai <pounjai at gmail.com> wrote:

> 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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>
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