<div dir="ltr">Hi all,<div> We have been lucky not to have the problem of carbon detaching from the grids. However of late, we are seeing residual substrate spanning the holes of both Quantifoil and C flat grids. This film is not visible under routineTEM analysis unless you add some kind of fiducial on them.We have started inspecting grids at random from a new box by adsorbing 3 microliters of colloidal gold on the grid. In most cases we are observing 10nm gold in the holes which shouldn't be the case if they are true hole. To our misfortune, we have over half dozen boxes with grids without true hole. </div><div>We have tried various solvent wash protocols, ethylene dichloride soak, ( Passmore & Russo, Methods Enzymol. 2016 technique) including the ones suggest by Quantifoil. The results have been highly variable depending on the batch of grids. Some clean reasonably well where as other batches of grids are quite resilient to even an over night soak in the solvents.</div><div>We have noticed the same kind of substrate even on the Aufoil grids.</div><div> </div><div>The question I have for the cryo EM community is whether any of you are facing this problem. More importantly, if you have a grid cleaning protocol that works well.</div><div><br></div><div>Thanks in advance for your valuable insight and suggestions</div><div><br></div><div>Vinod Nair,</div><div>Staff Scientist</div><div>NIAID/NIH</div><div><br></div><div><br></div></div><br><div class="gmail_quote"><div dir="ltr">On Fri, Dec 7, 2018 at 10:55 AM Briegel, A. <<a href="mailto:a.briegel@biology.leidenuniv.nl">a.briegel@biology.leidenuniv.nl</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">
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<div dir="ltr">Dear Cheri and Anna,
<div>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. <br>
I hope the problem can be solved.</div>
<div>Best wishes,</div>
<div>Ariane</div>
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<div id="gmail-m_-550301184325398581AppleMailSignature" dir="ltr">Sent from my iPhone</div>
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On 7 Dec 2018, at 18:26, Anna Sartori Rupp <<a href="mailto:anna.sartori.rupp@gmail.com" target="_blank">anna.sartori.rupp@gmail.com</a>> wrote:<br>
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<div dir="auto"><span style="font-family:sans-serif;font-size:12.8px">Hi Cheri,</span>
<div dir="auto" style="font-family:sans-serif;font-size:12.8px">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.</div>
<div dir="auto" style="font-family:sans-serif;font-size:12.8px">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.</div>
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<div dir="auto" style="font-family:sans-serif;font-size:12.8px">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. </div>
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<div dir="auto" style="font-family:sans-serif;font-size:12.8px"> 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. </div>
<div dir="auto" style="font-family:sans-serif;font-size:12.8px">It would be great if you could also contact them to report your problem.</div>
<div dir="auto" style="font-family:sans-serif;font-size:12.8px">I hope this helps,</div>
<div dir="auto" style="font-family:sans-serif;font-size:12.8px">Ciao </div>
<div dir="auto" style="font-family:sans-serif;font-size:12.8px">Anna</div>
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<div dir="auto" style="font-family:sans-serif;font-size:12.8px">Anna Sartori-Rupp , PhD</div>
<div dir="auto" style="font-family:sans-serif;font-size:12.8px">Ultrastructural Bioimaging Unit</div>
<div dir="auto" style="font-family:sans-serif;font-size:12.8px">Institut Pasteur,</div>
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<div dir="auto" style="font-family:sans-serif;font-size:12.8px"><span style="font-size:12.8px">75015 </span><span style="font-size:12.8px">Paris, France </span></div>
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<div dir="ltr">Il giorno Gio 6 Dic 2018, 9:12 PM HAMPTON, CHERI M CTR USAF AFMC AFRL/RXAS <<a href="mailto:cheri.hampton.ctr@us.af.mil" rel="noreferrer noreferrer" target="_blank">cheri.hampton.ctr@us.af.mil</a>> ha scritto:<br>
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<p style="margin-top:0px;margin-bottom:0px">Hi all,</p>
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<p style="margin-top:0px;margin-bottom:0px">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 :)<br>
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<p style="margin-top:0px;margin-bottom:0px">Thanks for your input,</p>
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<div>Cheri</div>
<div><br>
~'~'~'~'~'~'~'~'~'~'~</div>
<div>Cheri M. Hampton, PhD</div>
<div>Research Microscopist</div>
<div>UES, Inc.<br>
AFRL/RXAS<br>
2179 12th St, B652/R122<br>
Wright-Patterson AFB, OH 45433-7750<br>
(937) 255-9081<br>
<a href="mailto:cheri.hampton.ctr@us.af.mil" rel="noreferrer noreferrer noreferrer" target="_blank">cheri.hampton.ctr@us.af.mil</a>
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