[ccp4bb]: Oil and cryoprotectant protocol.

["Tom Caradoc-Davies" <toms_postingacct@hotmail.com>]

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Hi all,
       again sorry for the non-ccp4 post. I have recieved so many requests 
for this protocol that I thought it would be easiest to post this to the 
ccp4bb. If you are not interested in the combination of oil and 
cryo-solution then please skip this and my apologies! :)

For those who are interested here is the protocol:




OIL/CRYOPROTECTANT COMBINATION PROTOCOL:

Reagents:
Oil: I use bulk parrafin, LABCHEM brand by AJAX Chemicals.
If you use organics such as isopropanol in your mother liquor and this 
causes problems via entering the oil layer the oil can be pre-equilibrated 
by shaking it with mother-liquor.
Cryosolution: Your lab's standard cryo-solution for that particular xtal. I 
used mother liquor containing 30% glycerol.
Tools: Standard xtal handling loops (I use Hampton) that are a bit larger 
than you would normally use for a given xtal. This varies depending on xtal 
shape but the loop must form an oil film rather than pick up a blob of oil 
when removed from the oil layer.
Also normal plastic micro-bridges, xtal manipulation tools (cat whiskers, 
acupuncture needles etc.) coverslips and if required a skirt cut from a 
cryo-vial or a small clear plastic box large enough to hold a coverslip.

Protocol:
1.XTAL HARVERSTING:
Xtals can be harvested in the normal manner or under oil. If harvesting 
under
oil:
A. Sitting drop: layer 40-50microL of oil over the drop containg the xtal. 
The drop should move to the bottom of the micro-bridge (if not a touch with 
a whisker will move it). To stop a drop from drying it requires 2-3mm of oil 
between the top of the drop and the surface of the oil.
I use 2-6microL to grow xtals and with these drops 40microL of oil is enough 
when using a micro-bridge. If you use very large drop sizes you may require 
more oil or a larger well to accomodate this.
B. Batch method under oil: A micro-bridge has 40microL oil added to the 
well. The drop containing the xtals in the batch experiment is removed using 
a 10microL pipette tip and pipetted under the surface of the oil. To help 
prevent xtal loss take up a small volume of oil before and after the drop in 
the pipette
tip. If these extra oil drops do not merge with the oil in the micro-bridge 
a touch with an acupuncture needle between the oil/oil drop interface will 
collapse the oil drop into the oil layer.
C. Hanging Drop: Place the coverslip containg the xtal drop face up in a 
small clear plastic box and add sufficient oil to make a layer at least 
2-3mm between the top of the drop and the surface of the oil.
Alternatively use Martyn Symmons technique of cutting a section from a
cryo-vial and place this around the drop on the coverslip. Grease is used to 
seal the skirt to the coverslip and the skirt is filled with oil using a 1mL 
pipette.

2.XTAL TRANSFER TO CRYO-SOLUTION:
A second micro-bridge is clearly labelled and filled with 40microL oil and 
has a 6microL drop of cryo-solution added under the surface of the oil.
If this drop adheres to the surface of the drop or the sides of the well it 
can be moved to the bottom of the well using an acupuncture needle or 
whisker.
The xtal is looped out of the mother-liquor drop through the oil interface 
and into the cryo-drop in the second bridge. This is done in the usual 
manner.
Large xtals or those causing problems may require the loop to be held so 
that it is edge on to the viewer.

3. CRYO-COOLING:
After the xtal has equilibrated in cryo-solution or been moved sequentially 
through a required series of cryo-protectant concentrations it can be looped 
out as above.
If viewed under a microscope the loop should have a thin film of oil 
covering the span inside the loop (like the surface of a bubble). The xtal 
should be held in this film and only have a tiny amount of cryo-solution 
attached to the faces of the xtal. The xtal edges should be clear of 
cryo-protectant and extent into the oil film.
Due to the thin layer of oil coating the xtal it should be protected from 
drying out for at least 10 minutes. This allows for ample time to move the 
xtal to the goniometer and place it in the cryo-stream. The oil film does 
not seem to affect cryo-cooling (or annealing).

The two benefits of using this protocol are:
1. The xtal is totally protected from the air and this provides ample time 
for xtal handling or cryo-cooling.
2. The xtal is cryo-cooled in a far smaller volume of cryo-solution than 
that of normal methods and this produces a smaller diffuse solvent ring on 
the resulting X-ray image.

The use of oil to protect xtals from dehydration is commonly used during 
both harvesting and xtal transfer, and the only different step in this 
protocol is the combination of oil with cryo-protectant.
Thanks to Martyn Symmons for his advice and cryo-vial skirt trick, Kris Tesh 
for his great slide show with xtal tricks and tips, Stephan Ginell, Henry 
Bellamy, Harry Powell, Steve Ernst, Peter Moody and many others for your 
advice and comments.

Cheers,
       Tom




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