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--></style></head><body lang=EN-US link=blue vlink="#954F72"><div class=WordSection1><p class=MsoNormal>Dear Sergej,</p><p class=MsoNormal>I believe those are (almost) dried out buffer of your sample. They are black because of high concentration of salt. You observe this because you have carbon film, otherwise they will be just empty holes. </p><p class=MsoNormal>You should check your humidifier (less likely), or drastically reduce your blot time. If you are working with Vitrobot, and already have a very short blot time, say 1s, then you can try manual plunger, or concentrate your sample. Diluted samples are less viscous and thus easier to be blotted away. Also, the blot time should be much shorter when you have a carbon film. </p><p class=MsoNormal>Good luck!</p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>Best,</p><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>Xinghong Dai<br>Postdoctoral Scholar<br>Department of Molecular and Medical Pharmacology, UCLA<br>+1 310 447 4330</p><p class=MsoNormal><o:p> </o:p></p><div style='mso-element:para-border-div;border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal style='border:none;padding:0in'><b>From: </b><a href="mailto:Sergej.Masich@ki.se">Sergej Masich</a><br><b>Sent: </b>Wednesday, October 19, 2016 6:30 AM<br><b>To: </b><a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br><b>Subject: </b>[3dem] Weird droplets</p></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal>Dear List, <o:p></o:p></p><div><p class=MsoNormal><o:p> </o:p></p></div><div><p class=MsoNormal>I probably missed the discussion since many of you had observed this phenomenon I suppose (see attached images). This is a Quantifoil grid with additional continuous layer of thin (5 nm) carbon. The grid was glow-discharged during 120 seconds, the solution was applied to the “proper” side (facing grid-box centre), blotted and plunge-frozen in liquid ethane. Why do these droplets form? I do not see the droplets after applying solution to the opposite side of the grid. Shouldn’t the carbon surface <b><u>inside</u></b> the holes be homogeneously hydrophilic? The surface outside the holes seems to be completely hydrophobic. Is it due to residual plastic?<o:p></o:p></p></div><div><p class=MsoNormal><o:p> </o:p></p></div><div><p class=MsoNormal>Thank you in advance.<o:p></o:p></p></div><div><p class=MsoNormal><o:p> </o:p></p></div><div><p class=MsoNormal>Sergej<o:p></o:p></p><div><div><p class=MsoNormal style='margin-bottom:12.0pt'>=============================<br>Sergej Masich, Ph.D.<br>Dept. of Cell and Molecular Biology<br>Karolinska Institutet<br>171 77 Stockholm, Sweden<br>tel: (+)46 - 8 - 524 873 61<br>mobile: (+)46 - 736 - 833 693<br>e-mail: <a href="mailto:Sergej.Masich@ki.se">Sergej.Masich@ki.se</a><o:p></o:p></p></div></div><p class=MsoNormal><img border=0 width=836 height=881 style='width:8.7083in;height:9.177in' id="_x0035_2F18F01-AF4B-469D-BF0B-7C98EE65C883" src="cid:image003.jpg@01D22A39.AEFB1A40" alt="cid:86227476-7EB8-401D-8ECE-07FA4A306FC4"><o:p></o:p></p></div><div><p class=MsoNormal><img border=0 width=836 height=881 style='width:8.7083in;height:9.177in' id=FF7420D5-294B-4EF0-8C38-7AB19D14DA8F src="cid:image004.jpg@01D22A39.AEFB1A40" alt="cid:83003F53-9716-466E-BE17-51E2FD1AA1B4"><o:p></o:p></p></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><o:p> </o:p></p></div></body></html>