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Dan,</div>
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There are probably lots of ways to address this, but if you have access to a carbon coater, you can deposit a very light coating of carbon over your 2 nm layer. You obviously want the new layer to be as thin as possible, but that should be possible.<br>
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David Gene Morgan<br>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>From:</b> 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Dan Richman <d.e.richman@jhu.edu><br>
<b>Sent:</b> Wednesday, October 12, 2022 5:01 PM<br>
<b>To:</b> 3dem@ncmir.ucsd.edu <3dem@ncmir.ucsd.edu><br>
<b>Subject:</b> [External] [3dem] Hydration failure on continuous carbon despite solvent cleaning</font>
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Hi 3DEMers,</div>
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My lab had previous success with 2nm continuous carbon coated Quantifoils (QF R2/2 300 +2nm, or EMS cat # Q3100CR2-2NM), but hydration and particle adsorption stopped working when we got two new boxes (both are lot 201027). I tested the chloroform, acetone,
isopropanol cleaning procedure from Passmore and Russo 2016, checked by negative stain and a very stable control protein (confirmed to work on a CF400-UL grid with the same glow discharge and stain procedure). On the QF+2nm with or without cleaning, particles
aggregate in pooled stain patches (images attached). The failure in cryo-EM is similar, very dry squares, sometimes with central pools, but hardly any particles.<br>
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Has anyone seen this behavior with continuous carbon grids, especially the commercial coatings? Any recommendations for other cleaning techniques?</div>
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Unfortunately the glow discharge treatment has to be quite gentle otherwise the thin coating over the holes will break - we use 15 mA for 15 sec and then try to compensate with longer droplet incubation times, 20-60 seconds.</div>
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Thanks for any advice!<br>
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<span style="font-family:Arial,Helvetica,sans-serif"></span><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">Dan Richman, PhD</span><span id="x_ms-rterangepaste-start"></span><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">
</span><font size="2"><span style="font-size:8pt; font-family:Arial,Helvetica,sans-serif">(</span><font size="2"><span style="font-size:8pt; font-family:Arial,Helvetica,sans-serif">he/him/his</span></font><span style="font-size:8pt; font-family:Arial,Helvetica,sans-serif">)</span></font></font><span id="x_ms-rterangepaste-end"></span></font>
<div><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">Research Associate, Berger Lab</span></font></div>
<div><font size="2"></font><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">Dept of Biophysics and Biophysical Chemistry</span></font></div>
<div><font size="2"><span style="font-family:Arial,Helvetica,sans-serif">Johns Hopkins University School of Medicine</span></font>
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