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<p class="MsoNormal"><span style="font-size:11.0pt"> </span><o:p></o:p></p>
<p class="MsoNormal"><span style="font-size:11.0pt">We are giving the grid a small tilt bending it a little towards the blotting pad just after gripping it by the tweezers.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt">The full contact of the blotting paper with the grid pushes the grid back in place. </span><o:p></o:p></p>
<p class="MsoNormal"><span style="font-size:11.0pt"> </span><o:p></o:p></p>
<p class="MsoNormal"><u><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#002060">_ _ </span></u><o:p></o:p></p>
<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#002060">Dr. Gabriel A. Frank
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<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#002060">Structural Biology & Cryo-EM</span></b><o:p></o:p></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#002060">Department of Life Sciences and NIBN</span><o:p></o:p></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#002060">Ben-Gurion University of the Negev</span><o:p></o:p></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#002060">Office: 972-8-642-8621</span><o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:11.0pt"> </span><o:p></o:p></p>
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<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">From:</span></b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"> 3dem <3dem-bounces@ncmir.ucsd.edu>
<b>On Behalf Of </b>Daniel Asarnow<br>
<b>Sent:</b> Saturday, August 10, 2024 2:02 AM<br>
<b>To:</b> David Bulkley <davidbulkley@gmail.com><br>
<b>Cc:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> Re: [3dem] 3dem Digest, Vol 204, Issue 11</span><o:p></o:p></p>
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<p class="MsoNormal">Or one could 3D print a wider backing plate in nylon or ABS.<o:p></o:p></p>
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<p class="MsoNormal">-da<o:p></o:p></p>
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<p class="MsoNormal">On Fri, Aug 9, 2024 at 4:00<span style="font-family:"Arial",sans-serif"> </span>PM Daniel Asarnow <<a href="mailto:asarnow@msg.ucsf.edu">asarnow@msg.ucsf.edu</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal">I've always used the humidity at 100%, and watched closely to make sure the paper touches the grid, and then if the contact breaks early I give it another touch with the next spot of the paper before plunging. I also moved the blot position
in ~1 mm so that I get a mixture of good and hard contacts (which might still be good!) instead of a mixture of good and failed ones (which are definitely bad).<o:p></o:p></p>
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<p class="MsoNormal">I do think using a craft knife to cut a piece of foam to the right size and replacing the metal plate under the paper with that would solve the problem. It's the only difference from the Vitrobot in regards to this issue, that the paper
isn't backed all the way to the outer edge. There's a couple of screws that hold the plate in so I think it would be easy to do.<o:p></o:p></p>
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<p class="MsoNormal">-da<o:p></o:p></p>
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<p class="MsoNormal">On Fri, Aug 9, 2024 at 12:24<span style="font-family:"Arial",sans-serif"> </span>PM David Bulkley <<a href="mailto:davidbulkley@gmail.com">davidbulkley@gmail.com</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:1.0pt;color:white">It seems like humidity plays poorly with filter paper in the GP2 for most folks. We typically just leave our humidifier off when we are blotting cells. Is this a bad idea? We've been able to get
some decent lamella from cells blotted with</span><o:p></o:p></p>
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<p class="MsoNormal">It seems like humidity plays poorly with filter paper in the GP2 for most folks. We typically just leave our humidifier off when we are blotting cells. Is this a bad idea? We've been able to get some decent lamella from cells blotted
with the chamber humidifier off, but I'm curious if folks have done any proper testing on this to verify if the cells survive, and if they are damaged, what the symptoms are. It would suck to find out that, while my filter paper has been doing great, all
of my cells have been getting trashed. Curious if anyone has some real data on this.<o:p></o:p></p>
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<p class="MsoNormal">On Fri, Aug 9, 2024 at 12:00<span style="font-family:"Arial",sans-serif"> </span>PM <<a href="mailto:3dem-request@ncmir.ucsd.edu">3dem-request@ncmir.ucsd.edu</a>> wrote:<o:p></o:p></p>
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1. Re: Inconsistent Blotting with GP2 (Sen, Anindito)<br>
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Message: 1<br>
Date: Fri, 9 Aug 2024 17:14:26 +0000<br>
From: "Sen, Anindito" <<a href="mailto:andysen@tamu.edu">andysen@tamu.edu</a>><br>
To: Liz Montabana <<a href="mailto:liz.montabana@czii.org">liz.montabana@czii.org</a>>, "<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>"<br>
<<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>><br>
Subject: Re: [3dem] Inconsistent Blotting with GP2<br>
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HI Liz,<br>
<br>
Is the chamber humidity too high (the filter papers get wet?).<br>
<br>
Stay safe & Regards<br>
<br>
Anindito Sen. Ph.D<br>
<br>
Microscopy and Imaging Center<br>
Texas A&M University<br>
301 Old Main Dr<br>
College Station, TX 77843-2257<br>
<br>
Office: ILSB Room 1133<br>
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From: 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu">3dem-bounces@ncmir.ucsd.edu</a>> on behalf of Liz Montabana <<a href="mailto:liz.montabana@czii.org">liz.montabana@czii.org</a>><br>
Date: Wednesday, August 7, 2024 at 6:59 PM<br>
To: <a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a> <<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>><br>
Subject: [3dem] Inconsistent Blotting with GP2<br>
Hi All We are seeing a lot of inconsistent blotting with our GP2 plunge freezer (The longer we blot, the thicker our grids are, among other odd behavior) - and we wanted to ask others' experience to see if we could pinpoint any cause or<br>
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Hi All<br>
<br>
We are seeing a lot of inconsistent blotting with our GP2 plunge freezer (The longer we blot, the thicker our grids are, among other odd behavior) - and we wanted to ask others' experience to see if we could pinpoint any cause or feedback we could provide Leica
to enhance the performance.<br>
<br>
Please share your trade tips and secrets for wrangling the GP2 - We've had experience with several other GP2s and none of them shared this behavior. Our main samples are whole cells grown on grids.<br>
<br>
Thanks<br>
Liz Montabana<br>
Chan Zuckerberg Imaging Institute<br>
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