Hi all,
So I did what you recommended to me.
I looked at the grids on the T12 and took pics and then did a glow discharge.
After 2 mins we saw that most of the holes have broken carbon.
At 1 min we saw that there are no broken carbon. See the pic in the mail.
Thanks a lot,
Yehuda Halfon
________________________________________
From: Collaborative Computational Project in Electron cryo-Microscopy [[log in to unmask]] on behalf of Kyle Morris [[log in to unmask]]
Sent: Tuesday, July 24, 2018 4:56 PM
To: [log in to unmask]
Subject: Re: [ccpem] Broken continues carbon on girds.
Hi Yehuda,
Can you clarify, do you have continuous (continues?) carbon that you
floated onto these grids and what grid type are they (are we allowed
to discuss manufacturers on this list...)?
Two thoughts:
If you have a continuous carbon support then all of Bob Grassucci’s
comments apply. Otherwise if not, that edge of the hole that has the
broken carbon looks a lot like something we often see on 1.2/1.3 grids
from one of the big manufacturers, out of the box. You can check on a
screening scope at room temperature before you use the grid. It’s
something I wish the manufacturer would correct...
On the plus side, to me it looks like your protein is sitting in open
hole ice quite happily, so you a) don’t need a continuous carbon
support and b) assuming the rest of the hole looks like the bottom of
your attached image then you can just image away from the broken
carbon and not worry.
Best wishes,
Kyle
> On 24 Jul 2018, at 06:07, Tom Burnley - UKRI STFC <[log in to unmask]> wrote:
>
> Dear Bob,
>
>
> Many thanks for your helpful advice to Yehuda. One thing to note... 3DEM is a great resource but as far as the CCP-EM mailing list goes we're happy, and encourage, any EM related question not just those related to software.
>
>
> Cheers,
>
>
> Tom
>
> ________________________________
> From: Collaborative Computational Project in Electron cryo-Microscopy <[log in to unmask]> on behalf of Grassucci, Robert <[log in to unmask]>
> Sent: 24 July 2018 13:47:06
> To: [log in to unmask]
> Subject: Re: [ccpem] Broken continues carbon on girds.
>
> Hi Yehuda,
> The carbon can get broken in a few points in the preparation pipeline.
>
> 1. When you are picking up the thin carbon. Does it cover the whole grid, did it pop when you picked it up (not normally but it could happen).
>
> 2. When you glow discharge or plasma treat. If you use too much power or treatment time you can actually etch away the carbon or weaken it.
>
> 3. Blotting itself as you suspect can damage the carbon. You have tried the obvious settings force and time. Perhaps make the carbon slightly thicker. This will make the image noisier but better than no carbon.
> If you can make the sample more concentrated 5-10X eliminate the carbon. Any of the above points in the pipeline can be checked by putting the grid in a phase contrast light or the TEM prior to putting the sample on the grid. Good luck.
> Regards,
> Bob
>
> BTW. This question would be better suited for the 3DEM list server.
> 3DEM Mailing List [log in to unmask]<mailto:[log in to unmask]>
>
>
> From: Collaborative Computational Project in Electron cryo-Microscopy [mailto:[log in to unmask]] On Behalf Of Yehuda Halfon
> Sent: Tuesday, July 24, 2018 7:33 AM
> To: [log in to unmask]
> Subject: [ccpem] Broken continues carbon on girds.
>
>
> Hi there.
>
> For some reason, when we use the vitrobot we get broken or torn continues carbon on our grids, see the pic added.
>
> We get it on several blotting time and on blot force - 1. We do it at 100% humidity and 4 degrees.
>
> Did someone see this? Can it be fixed using different blotting conditions?
>
> Yehuda Halfon
>
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