[3dem] Inconsistent Blotting with GP2

Thu Aug 8 16:47:08 PDT 2024

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
when we were all doing manual plunge freezing. I am not sure the reason why
the GP2 blotting paper is so thin now...

Using two blotting papers on top of each other might do the trick, but I
have never tried that.
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.
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.

Hope this helps,
Best,
Sylvain

PhD
Research Fellow - Microscopy Australia

*Monash University*
Ramaciotti Centre for Cryo-EM
G92, 15 Innovation Walk, Clayton Campus
Victoria, 3800 Australia

E: sylvain.trepout at monash.edu

On Fri, 9 Aug 2024 at 03:33, Daniel Asarnow <asarnow at msg.ucsf.edu> wrote:

> 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.
>
> 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.
>
> Best,
> -da
>
> On Thu, Aug 8, 2024 at 2:16 AM Guillaume Gaullier <
> guillaume.gaullier at kemi.uu.se> wrote:
>
>> 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
>> ZjQcmQRYFpfptBannerStart
>> This Message Is From an External Sender
>> This message came from outside your organization.
>>
>> ZjQcmQRYFpfptBannerEnd
>> 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).
>>
>> I hope this helps,
>>
>> Guillaume
>>
>> Typed on a phone
>>
>> On 8 Aug 2024, at 07:20, Michael Elbaum <michael.elbaum at weizmann.ac.il>
>> wrote:
>>
>> 
>>
>> 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...
>>
>> regards,
>>
>> Michael
>> ------------------------------
>> *From:* 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Liz Montabana <
>> liz.montabana at czii.org>
>> *Sent:* Thursday, August 8, 2024 2:59:01 AM
>> *To:* 3dem at ncmir.ucsd.edu
>> *Subject:* [3dem] Inconsistent Blotting with GP2
>>
>> *Caution: External Sender. Do not click on links or open attachments
>> unless you recognize the sender.*
>>
>> 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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