Checking Cryo-Conditions under the microscope can actually be very
useful. Even if you have access to a X-ray beam, simple pre-tests can
be very informative.
This can be done as follows:
A 24well plate cover is placed on a stereo microscope open side down,
a second one on top of it the other way around. Carefully pour lN2
into it (might make a crackling noise, however the plastic should
stand it). A 1 mm loop is placed on top of a 'crystal wand' tool,
dipped first into the cryo buffer under investigation and then quickly
into the lN2 on the microscope. Hold the loop right under the lN2
surface and focus. Vitrification of the shock-cooled buffer can now be
assesed.
I usually check a series with increasing concentrations of
cryo-protectant to determine the critical point for successful
vitrification. To the final buffer I then add a certain safety margin
to avoid unpleasant surprises during the synchrotron trip, if an X-ray
test is not possible.
Clemens
Zitat von Karthik S <biokart...@gmail.com>:
I have not heard of anyone checking for suitable cryo in that fashion how
fast can you do it before it is affected by room temp and how would you know
about the ice rings, but when you have crystal(s) and not crystal you can
freeze different ones in several different cryos (sugars, glycerol etc) and
take them with you to the trip. good luck!
--
Karthik
Graduate Student-Biophysics
University of Michigan
Ann Arbor, MI 48109
karth...@umich.edu
734-763-3384
On Tue, Sep 22, 2009 at 4:24 AM, Claudia Scotti
<claudiasco...@hotmail.com>wrote:
Dear List,
Sorry for the probably silly question.
Any suggestions to test cryoconditions without X-rays or cryostream?
I'd need to freeze crystals before going to ESRF and I'm a bit anxious. Is
it enough to try to freeze the cryoconditions in liquid nitrogen checking
under the microscope (or by eye) or is this still risky?
Thanks,
Claudia
Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia
Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039
0382 986335/8/1 Facs 0039 0382 303673
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