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Here is a summary of previous messages regarding ice from the SSRL mailing list. Kendall ---------------------------------------------------------------------------- Our "innovation" has been to keep all of the ports of the cassette filled with "blank" pins (we color the bases red with a Sharpie marker so we can tell them apart), and then only un-plug a row of ports at a time for filling with crystals. We've found that this prevents floating ice particles from settling into the empty ports and turning our freshly loaded samples into snowballs. This has reduced our icing issues by >>90%. I'd say that it is close to a 100-percent solution. The lesson for us seems to be that the "icy-ness" of the liquid nitrogen doesn't appear to be the critical factor in terms of freezing the sample (and the resultant icy-ness), but that allowing the ports to fill with snow is nearly always highly detrimental! -Tom --- Thomas J. Stout Hello Ana et al., A way that has been very successful for us in preventing ice from accumulating in the liquid nitrogen and then getting into the cassette ports, is to preload the cassette with empty pins. This is a tip coming from an SF based Biotech. You then just have to remove the empty pin right before loading your flash frozen crystal, hence, no ice on your crystal as long as you don't too much ice floating around. Cheers Steph Just to add my 2 cents (from SGPP time) We premounted our crystals in canes and stored them in a LN2 dewar. Once we were ready to ship we would get all our crystals (somewhere between 200 - 400) and transfer them from the canes into the cassettes. One cassette took roughly 15 -20 minutes, depending who would do the transfer. Our failure rate due to ice during transfer has been zero. If we had ice, then it was more a cryo-issue and the fault of the user not having the right amount of cryo-protectant. We once had a Fedex problem, where we believe the cassettes were taken out and "inspected" all crystals of the top cassette were essentially dead or in pretty bad shape. When we transfer the crystals, we have three persons involved. One who drops the cane mounted crystal into a small bowl filled with LN2, the mounter (the one who is to blame if something goes wrong :-) ) and somebody who takes notes which crystals was transferred into which port. We also keep adding LN2 during the process of transferring the crystals, furthermore we precool the cassettes in a different cane dewar before transferring it into the blue box. And I don't use the mounting device, sorry. Some of us do, but I prefer to go with the Hampton magnetic wand - it's just much faster to handle the samples. Juergen Hi Everyone, Great job setting this email list up Ana. To echo Aina and the presentations from the recent remote, remote, data workshop we've come up with several things that seem to work for us on the icing. We fill in a fume hood which draws the nitrogen boil off up in a stream. This seems to prevent air and the water vapor it contains getting anywhere near the fill station. In this case we avoid using the blue cover and use the fume hood sash quite high up. We use two tool sets and have one warming while the other is in use (swap about every 4 samples). Before using this we had ice rapidly becomming evident in the fill station resulting in several emptying, drying and refilling steps. Now we can fill a whole cassette without seeing any serious ice. It helps to use a microscope illuminator directly into the fill station both for filling and to detect any initial ice build up. Before transfering the cassette to the dewar we remove any ice from the transfer tool then as we lift the cassette we wash it by pouring liquid nitrogen over it, similar to removing ice from a crystal. This seems to help quite a lot and has greatly improved our icing problems from the initial runs. Using vials and canes has worked very well for one of my collaborators and I will be trying that in the future. It seems a very rapid way of filling the cassette and speed is a key factor to prevent icing problems. Ana mentioned Robert Thornes work. Basically Thorne points out for nitrogen boil off there is a significant temperature gradient from the liquid up. By blowing away the boil off the temperature transition is sharp and therefore cooling more rapid. This suggests why freezing in the stream might be better than plunging as you have a much sharper temperature transition. You can also take a nice snapshot of the crystal before it leaves home. I think the result can also be adapted to the plunging technique by using a ambient gas stream within the guide tool. We'll be playing with this during the shutdown. Lab motto, "Ice is our enemy" Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: [EMAIL PROTECTED] Telepathy: 42.2 GHz Heisenberg was probably here! > Hi Everyone, > > Over the next two weeks, I will put together some new web-based directions > on cassette loading that will include a lot of helpful information on how > to reduce ice. These webpages will include material from the remote data > collection workshop presentations. > > > Thanks, > > Aina > > > > ________________________________ > > From: [EMAIL PROTECTED] on behalf of Mathews, > Irimpan > Sent: Mon 8/7/2006 1:40 PM > To: Gonzalez, Ana M.; [EMAIL PROTECTED] > Subject: Re: [Px-ssrl] Ice formation on crystals > > > > > Hi, > > If I take long time (to fish them/other cryo steps) to mount the crystals, > I usually rotate the cassette before putting the next crystal. This seems > to help with removing ice flakes that are accumulated inside the ports > that are on the top side. > > Mathews > > -----Original Message----- > From: [EMAIL PROTECTED] > [mailto:[EMAIL PROTECTED] On Behalf Of Ana Gonzalez > Sent: Monday, August 07, 2006 11:04 AM > To: [EMAIL PROTECTED] > Subject: [Px-ssrl] Ice formation on crystals > > > A couple of groups have been asking about how to prevent ice flakes on the > crystals when transferring them to the SAM cassette. > There are two practices which are critical to avoid ice: > > 1) Use the lid to cover the box when you are not mounting crystals. To > prevent ice from accumulating in the liquid nitrogen and getting into the > cassette ports, the cassette should not be stored in the loading dewar for > more than 20 minutes. > > 2) Plunge the crystal as fast as possible into the liquid nitrogen. > > Recently, Robert Thorne's group has shown that blowing away the cold gas > layer above the liquid nitrogen while freezing the crystal minimizes ice > formation; they use dry air flowing through a 10mm nozzle directed at the > liquid at a shallow angle. They presented this work at the ACA meeting and > it should be published soon. > Although they refer mainly to crystalline ice within the sample volume > rather than ice flakes attached to the sample, this idea makes a lot of > sense, so I though I'd pass the information along. > > Ana > -- > ------------------------------------------- > Ana González <[EMAIL PROTECTED]> > Staff Scientist > Stanford Synchrotron Radiation Laboratory > 2575 Sand Hill Road, MS 99 > Menlo Park CA 94025 > Phone: (650) 926 8682 Fax: (650) 926 3292 > ------------------------------------------- > > _______________________________________________ > Px-ssrl mailing list > [EMAIL PROTECTED] > http://smb.slac.stanford.edu/mailman/listinfo/px-ssrl > > > > _______________________________________________ > Px-ssrl mailing list > [EMAIL PROTECTED] > http://smb.slac.stanford.edu/mailman/listinfo/px-ssrl > _______________________________________________ Px-ssrl mailing list [EMAIL PROTECTED] http://smb.slac.stanford.edu/mailman/listinfo/px-ssrl On 11/1/06 5:29 PM, "Chunmin L" <[EMAIL PROTECTED]> wrote: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > Hi, > > We are preparing a remote data collection at SSRL. Last time, we had > icing problem when we load mounted crystals into the sample cassette. > Any tips to reduce or prevent it are very welcome. > > Thanks in advance! > > > Chunmin
