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Dear Rebecca,
If everything fails and you have few more drops of crystals and
do not know how to freeze mount, here is another way you can try :-
If you have few crystals in the drop under oil and the drop size
(excluding oil!!) is few microliters: add 10 microlitre of mother liquor
(you can get the mother liquor conc. based on few trials; it should be few
% more than the final conc. of the one you had used while setting up the
microbatch, a decent start will be 5 % more ), allow it to stand for few
minutes. Then use the classical cappillary mounting method to suck the
crystals slowly out and onto a cover slip. If possible try to remove as
much as possible (you may not be able to remove everything) the
halo of oil surrounding the drop on cover slip (using the same capillary
watching under microscope to make sure you are not sucking out the
crystals). The use of mother liquor (the 10 ul) is to basically to
reduce the oil that come along when you suck the crystals, so if necessary
you can use more ul's.
Now quickly scoop the crystal with a loop and dip in the
cryoprotectant and freeze it, in the usual way. This has worked for me,
though it may need some more standardization depending on your case.
HTH,
Natesh
On Wed, 13 Sep 2006, vetting wrote:
> *** For details on how to be removed from this list visit the ***
> *** CCP4 home page http://www.ccp4.ac.uk ***
>
>
>
>
> Rebecca,
>
> I think the success of mounting out of microbatch depends on the type of
> oil and plate you are using. We successfully are able to transfer crystals
> out of drops and into cryoprotectants using a standard hampton mounted
> cryoloop. You need a microscope that has a decent distance between the
> plate and actual lens apparatus so you can get the cryoloop down far
> enough into the well without covering up your viewpoint. When the drop
> contains the cryoprotectant already... then we directly transfer to liquid
> nitrogen and do not try and remove the oil.... at that point the oil only
> covers the solution which surrounds your crystal. One of the main benifits
> of vapour diffusion under oil vs. other sitting drop type methods is that
> the crystal (at least in our arrangement/apparatus) never sticks to the
> bottom, since the drop is "spericilized" by the oil and barely touches the
> plastic. Go to
> http://www.bioc.aecom.yu.edu/labs/blanlab/VETTING/PROTOCOLS/vapdiffunderoil.html
> if you want to check out our VDUO setup.
>
> Matthew Vetting
>
> On Tue, 12 Sep 2006, Page, Rebecca wrote:
>
> > *** For details on how to be removed from this list visit the ***
> > *** CCP4 home page http://www.ccp4.ac.uk ***
> >
> >
> > CCP4 community:
> > I find mounting crystals grown in microbatch drops difficult compared to
> > mounting crystals grown out of sitting drops, primarily because of the oil
> > layer. Do most people simply use the oil layer as a cryoprotectant? If not,
> > do you typically try to remove the oil prior to transferring the crystal to
> > a cryoprotectant. I'd like to compile 'tricks of the trade' for mounting
> > crystals out of microbatch plates. Any advice would be helpful and I'll
> > compile the list of responses and repost for the community.
> >
> > Thanks in advance,
> >
> > Rebecca
> >
>
