Rather late in the day, I second all these suggestions, but would like to add that it is also important to change the liquid nitrogen in the small Dewar into which you are cooling (not freezing* I hope!) your crystals. If you are cooling a lot of crystals sequentially, taking the lid off and on many times adds to the chance of ice floating around in the Dewar and getting onto the loops. I usually have 2 foam Dewars, one in use and one warming up ready for new nitrogen after I have cooled a maximum of 10 crystals in one batch of Liq nit.
Best wishes
Elspeth
* 'freezing' implies a phase change, and that is exactly what we DON'T want to happen...
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Sergei Strelkov
Sent: 12 August 2015 10:54
To: [log in to unmask]
Subject: Re: [ccp4bb] Ice on loops?
I wanted to thank everyone for very insightful replies to my question!
To summarize the most important points:
1. Ice in the LN2 reservoir can be a problem.
Several people report that the source of the LN2 matters.
I was impressed by Ilme's advice to use coffee filters (we'll try this!)
2. Care should be taken to minimize ice formation during freezing. Humidity in the room should be kept low and the LN2 vessel should be covered all the time.
3. One can try to remove the ice from loops using a
LN2 'jet' (we tried this before with some success) or a fine paintbrush.
Trying to freeze as quickly as possible is certainly a good idea, but in our limited experience this mostly does not resolve the problem of ice formation on the surface...
Many thanks again,
Sergei
On 11-Aug-15 3:16 PM, Ilme Schlichting wrote:
> Hello Sergei,
>
> we often filter the liquid nitrogen before we use it. We use
> old-fashioned funnel-shaped paper filters in a plastic funnel, pretty
> much like making coffee. I think this also removes the tiny ice seeds.
>
> Good luck
> ilme
>
> On 8/11/2015 2:21 PM, Sergei Strelkov wrote:
>> Dear All,
>>
>> What I wanted to discuss specifically is the ice crystals that we
>> often find on the outside of the loops rather than bulk ice formation
>> which is due to insufficient cryoprotection (although it is sometimes
>> difficult to distinguish between the two cases). Sometimes these ice
>> crystals on the surface can be removed by pouring LN2 onto the loop,
>> but more often this has little effect.
>> 'Annealing' (thawing and freezing again) always resolves the problem
>> but this is of course not an option most of the time (as the protein
>> crystal gets damaged).
>>
>> We measure at synchrotrons on a regular basis and the ice formation
>> problem seems to vary quite a bit from trip to trip. Thus far we
>> could not quite figure out what causes it.
>>
>> We usually mount our crystals (with cryoprotectant...) by plunging
>> them into LN2 on a bench. Thereafter they are stored either on canes
>> or unipacks in a dry shipper. As we travel by car or train, we always
>> keep the dry shipper filled with LN2 anyway. At the beamline they are
>> usually mounted by a robot.
>>
>> We mostly use the fiber loops (Hampton) but sometimes also the 'plastic'
>> loops
>> from Mitegen. I could not see much difference between them in terms
>> of ice formation, but I wondered what the others' experiences are.
>>
>> The choice of cryoprotectant matters obviously but annoyingly we can
>> sometimes happily use some cryoprotecting conditions initially, but
>> suddenly have problems with exactly the same crystals and
>> cryoprotectant on the next trip...
>>
>> The ice accumulation in any one of the LN2 vessels used at different
>> stages
>> (LN2 tank, the open reservoir we use for plunge-freezing, or the
>> crystal storage/shipping dewar) /might/ be the cause. Specifically,
>> there will /always/ be /some/ ice accumulation in the open reservoir.
>> Thus far we were not too paranoic about refreshing LN2 every five
>> minutes -- but maybe we should?...
>>
>> Or could the (variable) presence of ice in the 100l LN2 tank that our
>> internal services fill for us be a problem?...
>>
>> Thanks and best wishes,
>> Sergei
>>
>> --
>> Prof. Sergei V. Strelkov
>> Laboratory for Biocrystallography
>> Dept of Pharmaceutical and Pharmacological Sciences, KU Leuven
>> Herestraat 49 bus 822, 3000 Leuven, Belgium
>> Work phone: +32 16 330845 Mobile: +32 486 294132
>> Lab pages:http://pharm.kuleuven.be/Biocrystallography
>>
>
--
Prof. Sergei V. Strelkov
Laboratory for Biocrystallography
Dept of Pharmaceutical and Pharmacological Sciences, KU Leuven Herestraat 49 bus 822, 3000 Leuven, Belgium
Work phone: +32 16 330845 Mobile: +32 486 294132
Lab pages: http://pharm.kuleuven.be/Biocrystallography
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