<div dir="ltr"><div>Hi Sylvain,</div><div>I think the pre-punched papers are Whatman 595 which is indeed quite thin. I've found more consistent behavior from the thicker No. 4's (or No. 1's), which you can punch out yourself using e.g. the very nice CNC milled punches from SubAngstrom. When only 595s are available I like to stack two of them, which fits on either the Vitrobot or GP2.<br></div><div><br></div><div>If you punch your own filters, you can also choose an ashless variety and avoid any trace Ca2+.<br></div><div><br></div><div>Best,</div><div>-da<br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Thu, Aug 8, 2024 at 4:47 PM Sylvain Trepout <<a href="mailto:sylvain.trepout@monash.edu">sylvain.trepout@monash.edu</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"><div class="msg-6119984403244748535">
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Hi there GP2-enthusiasts, In our GP2 we use the classic Ted Pella blotting paper (prod. number 47000-200), and it is quite thin, much thinner than what we use in our Vitrobots for instance, and much thinner than what we used back in the days</div>
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<div dir="ltr">Hi there GP2-enthusiasts,<div><br></div><div>In our GP2 we use the classic Ted Pella blotting paper (prod. number 47000-200), and it is quite thin, much thinner than what we use in our Vitrobots for instance, and much thinner than what we used back in the days when we were all doing manual plunge freezing. I am not sure the reason why the GP2 blotting paper is so thin now...</div><div><br></div><div>Using two blotting papers on top of each other might do the trick, but I have never tried that.</div><div>What we installed on our GP2 is some kind of plastic washer (it is transparent and flexible and we manually cut it to the dimensions of the blotting paper) that we put at the back of the blotting paper, and it helps prevent the filter paper from getting all wavy when the humidity in the chamber is high.</div><div>However, when the filter paper stays inside the humid chamber for a fair amount of time, the wavyness cannot be avoided, so we simply change it for a new one, even if we did not use it 10 times.</div><div><br></div><div>Hope this helps,</div><div>Best,</div><div>Sylvain</div><div><div><div><div><div dir="ltr" class="gmail_signature"><div dir="ltr"><p style="color:rgb(34,34,34);margin-bottom:0px"><span style="color:rgb(80,80,80);font-family:sans-serif">PhD</span><br>Research Fellow - Microscopy Australia</p><p style="color:rgb(34,34,34);margin-bottom:0px"><b>Monash University</b><br></p><span style="color:rgb(34,34,34)">Ramaciotti Centre for Cryo-EM</span><br style="color:rgb(34,34,34)"><span style="color:rgb(34,34,34)">G</span><span style="color:rgb(34,34,34)">92, 15 Innovation Walk, Clayton Campus</span><br style="color:rgb(34,34,34)"><span style="color:rgb(34,34,34)">Victoria, 3800 Australia</span><p style="color:rgb(80,80,80);font-family:sans-serif;margin-bottom:0px">E: <a href="mailto:sylvain.trepout@monash.edu" style="color:rgb(17,85,204)" target="_blank">sylvain.trepout@monash.edu</a></p></div></div></div><br></div></div></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Fri, 9 Aug 2024 at 03:33, Daniel Asarnow <<a href="mailto:asarnow@msg.ucsf.edu" target="_blank">asarnow@msg.ucsf.edu</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"><div dir="ltr"><div>I've noticed it's very common for the paper to break contact with the drop at the end of blotting, and that the paper tends to wrinkle due to the humidity in the chamber, causing the actual force to vary wildly between blots. You can try giving the grid a 2nd short blot, as well as stiffening the paper with a piece of tape or by stacking two of them.</div><div><br></div><div>A modification I have been planning on testing is to add a piece of thin foam cut to a larger size behind the paper, to provide some backing like on the vitrobot.<br></div><div><br></div><div>Best,</div><div>-da<br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Thu, Aug 8, 2024 at 2:16 AM Guillaume Gaullier <<a href="mailto:guillaume.gaullier@kemi.uu.se" target="_blank">guillaume.gaullier@kemi.uu.se</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"><div>
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If you are using the blot sensor, and it doesn’t detect correctly, then it cancels the blot (after having touched the grid for a bit) but still plunges, and the resulting ice is much thicker than the desired result. Normally you should get a</div>
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If you are using the blot sensor, and it doesn’t detect correctly, then it cancels the blot (after having touched the grid for a bit) but still plunges, and the resulting ice is much thicker than the desired result. Normally you should get a message on the
screen if this is the problem, but it’s possible that the blot sensor is misbehaving on your unit (the only Leica EM GP2 I used around 2017-18 had a very unreliable blot sensor, so I am biased from this experience; maybe newer units don’t have this problem
anymore).
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<div>I hope this helps,</div>
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<div dir="ltr">Typed on a phone</div>
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<blockquote type="cite">On 8 Aug 2024, at 07:20, Michael Elbaum <<a href="mailto:michael.elbaum@weizmann.ac.il" target="_blank">michael.elbaum@weizmann.ac.il</a>> wrote:<br>
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<p>Hi Liz, something that comes to mind is a fault that maybe the blotting paper doesn't turn. You end up blotting with the same wet area. (yes, this happened to me and I didn't pay attention till too late) You can mark the paper and just run it through
the motions without a tweezer, to make sure it turns on every cycle. Something to rule out, at least...</p>
<p>regards,</p>
<p>Michael<br>
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<b>Sent:</b> Thursday, August 8, 2024 2:59:01 AM<br>
<b>To:</b> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
<b>Subject:</b> [3dem] Inconsistent Blotting with GP2</font>
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<div dir="ltr">Hi All
<div><br>
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<div>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.</div>
<div><br>
</div>
<div>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.</div>
<div><br>
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<div>Thanks</div>
<div>Liz Montabana</div>
<div>Chan Zuckerberg Imaging Institute</div>
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