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[ccp4bb]: New xtal handling method?
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I think it is always worth posting tricks and tips. I have often
mounted crystals in small _hanging_ droplets by making an 'oil-bath'
around them. You cut off the skirt of a cryovial and stick it onto the
coverslip as soon as you open the well and flip over the coverslip. I use
grease to seal it to the coverslip and then flood the area around the
droplet with oil. I usually use Al's oil, others would work better
probably - there are always leaks but you can top it up and then work on
the droplet at your 'leisure'. Good if you need to do surgery or a long
drawn out cryo-protection scheme - always sooo relaxing of course.....
good luck with all your crystals
On Wed, 29 Jan 2003, Tom Caradoc-Davies wrote:
> Date: Wed, 29 Jan 2003 02:28:24 +0000
> From: Tom Caradoc-Davies <firstname.lastname@example.org>
> To: email@example.com
> Subject: Re: [ccp4bb]: New xtal handling method?
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> *** CCP4 home page http://www.ccp4.ac.uk ***
> Hi all,
> thanks for the Emails! I should have been a bit more specific.
> Thanks for the info that that oil has been used for xtal mounting over the
> years (esp Paratone) and it's use is common in small molecular xtallography.
> What I was wondering about is do people routinely use oil to protect xtals
> (produced by normal vapour diffusion expts) on loops from drying during
> transit from cryo-solution to cryo-stream?
> I'm sure we are all familiar with the "get the loop containing the xtal from
> the cryosolution to the cryostream before it dries out" game and have seen
> xtals lost/damaged in the rush (well in my hands anyway!).
> Using a thin film of parrafin oil a xtal can be looped out of cryo-solution
> and is protected for at least 10 mins. So instead of the rush you could have
> as much time as you want to move from the microscope to the goniometer and
> get the xtal aligned in the cryo-stream.
> I tested this by exposing an xtal to X-rays while it was in a loop covered
> by a film of oil (no capillary or cryo-stream to protect it) and this
> protected it from dehydration for over 45 minutes.
> Micro-bridges filled with 40microL of paraffin oil and containing a 6microL
> drop of cryo-solution full of nice xtals were left uncovered for a week on a
> microscope stage and after that time xtals still diffracted as well as
> controls. This is a real help for me as I can screen a range of xtals from
> different expts without having to open and close vapour diffusion expts or
> rush because 1microL drops are drying out while I'm trying to loop out the
> best xtal.
> I am suggesting combining the use of a cryo-solution with oil rather than
> the technique where the xtal is placed in oil and a wick is used to remove
> mother liquor from the xtal. Oil is layered over the drop from the vapour
> diffusion experiment and then the xtal looped into a drop of cryo-solution
> under 40microL of paraffin oil in a micro-bridge. If you have to move a xtal
> through a series of cryoprotectant concentrations, placing paraffin oil over
> the sitting drops allows the xtal to be protected from drying.
> If you look at the loop under a microscope the xtal has a thin film of
> cryo-solution coating the planes of the xtal and this is held in the oil
> film. Using this technique there is a lower diffuse scatter on X-ray images
> than using normal xtal handling methods and I assume that this is due to
> less volume of cryo-solution on the xtal than a normal drop and the paraffin
> oil not producing much diffuse scatter but this needs more research. Oh and
> I use a slightly larger loop than normal to get a thin film of oil and less
> cryo-solution on the xtal.
> I guess I just found this trick to be so incredibly useful that I wondered
> if it could be of use to anyone else?
> Again if this is old hat I apologise and sorry about the length of this
> Tom Caradoc-Davies
> Biochem Dept
> Univ. Otago
> New Zealand
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Martyn F. Symmons
Crystallography and Biocomputing Group
Department of Biochemistry
University of Cambridge
Phone: 01223 766020
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