[3dem] 3dem Digest, Vol 204, Issue 11

Sat Aug 10 07:35:30 PDT 2024

We are giving the grid a small tilt bending it a little towards the blotting pad just after gripping it by the tweezers.
The full contact of the blotting paper with the grid pushes the grid back in place.

_                                                                _
Dr. Gabriel A. Frank
Structural Biology & Cryo-EM
Department of Life Sciences and NIBN
Ben-Gurion University of the Negev
Office: 972-8-642-8621
Lab: 972-8-642-8852
Building 41/128
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From: 3dem <3dem-bounces at ncmir.ucsd.edu> On Behalf Of Daniel Asarnow
Sent: Saturday, August 10, 2024 2:02 AM
To: David Bulkley <davidbulkley at gmail.com>
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] 3dem Digest, Vol 204, Issue 11

Or one could 3D print a wider backing plate in nylon or ABS.

-da

On Fri, Aug 9, 2024 at 4:00 PM Daniel Asarnow <asarnow at msg.ucsf.edu<mailto:asarnow at msg.ucsf.edu>> wrote:
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).

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.

-da

On Fri, Aug 9, 2024 at 12:24 PM David Bulkley <davidbulkley at gmail.com<mailto:davidbulkley at gmail.com>> wrote:
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
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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.

On Fri, Aug 9, 2024 at 12:00 PM <3dem-request at ncmir.ucsd.edu<mailto:3dem-request at ncmir.ucsd.edu>> wrote:
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Today's Topics:

   1. Re: Inconsistent Blotting with GP2 (Sen, Anindito)

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Message: 1
Date: Fri, 9 Aug 2024 17:14:26 +0000
From: "Sen, Anindito" <andysen at tamu.edu<mailto:andysen at tamu.edu>>
To: Liz Montabana <liz.montabana at czii.org<mailto:liz.montabana at czii.org>>, "3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>"
        <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Subject: Re: [3dem] Inconsistent Blotting with GP2
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HI Liz,

Is the chamber humidity too high (the filter papers get wet?).

Stay safe & Regards

Anindito Sen. Ph.D

Microscopy and Imaging Center
Texas A&M University
301 Old Main Dr
College Station, TX 77843-2257

Office: ILSB Room 1133
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From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Liz Montabana <liz.montabana at czii.org<mailto:liz.montabana at czii.org>>
Date: Wednesday, August 7, 2024 at 6:59 PM
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu> <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Subject: [3dem] Inconsistent Blotting with GP2
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
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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 feedback we could provide Leica to enhance the performance.

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.

Thanks
Liz Montabana
Chan Zuckerberg Imaging Institute
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