[3dem] carbon detaching from grids

[Bowman, Valorie D]

Folks,


I experienced this as well a couple of years ago- even having some grids BAD within a box and others ok!  It's especially bad with peeling off &/or breakage of the film if you are using 200 mesh grids: all that open space.


It seemed to correlate with the color of the carbon side.  If it was black = the carbon film was strong, if it was light or nearly invisible, i.e. gold or copper colored, ->  broken films everywhere!  I even tried the "thick" films. They were just as inconsistent. Some were black and strong, some were shiny and weak.


To save heart break, I started screening them and only use the black ones "as is".  The shiny one I evaporate a thick layer of carbon over the supposed C side, then they work fine too.


I know, it's a pain to have to re-work something you bought to save time, but it's better than sending them back and maybe waiting 6 months for a replacement!


Let me know if this works for you !   :)



Cheers,

Valorie

Valorie Bowman

vdb at purdue.edu
765-494-5643
EM Facility Laboratory Manager/
Senior Research Electron Microscopist
Purdue Cryo EM Facility
Purdue University
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Briegel, A. <a.briegel at biology.leidenuniv.nl>
Sent: Friday, December 7, 2018 12:55 PM
To: anna.sartori.rupp at gmail.com
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] carbon detaching from grids

Dear Cheri and Anna,
We also had this problem recently with our standard samples (with same glow discharger, plunge freezer and same buffers), so it might  indeed be related to the new manufacturing process.
I hope the problem can be solved.
Best wishes,
Ariane


Sent from my iPhone

On 7 Dec 2018, at 18:26, Anna Sartori Rupp <anna.sartori.rupp at gmail.com<mailto:anna.sartori.rupp at gmail.com>> wrote:


Hi Cheri,
We recently experienced exactly the same problem with the two latest purchased boxes of quantifoil gold R2/2 grids. We used the same glow discharge machine (ELMO) and the same settings that we normally use and we experienced that when we put the grids in a buffer like PBS or HEPES + 2% PFA the carbon was detaching on almost all the grids.
To see where the problem could come from we used two very old batches of quantifoil grids and repeated the same procedure , but in this case the carbon remained nicely attached to the grids.

So it could be that your problem might not be due to your plasma cleaner but rather to the new way quantifoil is manifacturing their grids.

 I contacted quantifoil and sent them the corresponding pictures, they told me that in fact they recently changed their carbon film deposition parameters (he talked about different plasma cleaner conditions...) and they told me that they would look into it , but I haven t heard back from them yet.
It would be great if you could also contact them to report your problem.
I hope this helps,
Ciao
Anna

-------------------------------------------------

Anna Sartori-Rupp , PhD
Ultrastructural Bioimaging Unit
Institut Pasteur,
28 rue du Dr Roux
75015 Paris, France


Il giorno Gio 6 Dic 2018, 9:12 PM HAMPTON, CHERI M CTR USAF AFMC AFRL/RXAS <cheri.hampton.ctr at us.af.mil<mailto:cheri.hampton.ctr at us.af.mil>> ha scritto:

Hi all,


I have suddenly begun experiencing the carbon detaching from my QF R2/1 grids when submersed in buffer and when applying proteins for blotting and cryo-plunging. I have tried two different lots of grids, and now I am suspicious of the facility's plasma cleaner that I have been using to glow discharge. All settings and timings have remained constant on my part. I plan to seek out another plasma cleaner to do a comparison test, but meanwhile, what kinds of solvents or materials placed in the plasma cleaner/glow discharge unit might linger and cause problems? I'd like to justify having a "clean" one just for TEM :)


Thanks for your input,

Cheri

 ~'~'~'~'~'~'~'~'~'~'~
Cheri M. Hampton, PhD
Research Microscopist
UES, Inc.
AFRL/RXAS
2179 12th St, B652/R122
Wright-Patterson AFB, OH 45433-7750
(937) 255-9081
cheri.hampton.ctr at us.af.mil<mailto:cheri.hampton.ctr at us.af.mil>


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