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[ccp4bb]: Summary of oil and cryo-protectant combo.
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as per normal here is a summary of the emails etc about the use of
oil and cryo-protectant together:
The technique described was placing a layer of paraffin oil over a drop
containing xtals in cryo-solution so that once the xtal is looped out
through the oil a thin oil film holds the xtal in the loop and prevents
dehydration. This film provides at least 10 minutes for the xtal to be
placed in the cryo-stream. The use of oil also produces a smaller volume of
cryo-protectant, as this clings to the faces of the xtal leaving the edges
poking into the oil layer, and a resulting smaller diffuse solvent ring.
1. It seems that while the use of oil for mounting xtals is common, labs
tend to use either oil OR a cryoprotectant for a particular xtal. If people
do combine the two together as I described above this method does not seem
to be commonly used (unless I misunderstood Stephan Ginell's Email!). :)
2. Martyn Symmons has a nice trick of making a skirt out of a cryo-vial and
sealing this with grease to make a "well" around a drop on a coverslip which
can be filled with oil. This prevents the drop from drying out allows more
time to loop out the xtal.
3. Kris Tesh, Peter Moody, Harry Powell, Stephan Ginell and Henry Bellamy
let me know that oil is used in many labs for several steps in xtal growth,
mounting and by itself as a cryo-protectant.
4. Several of the above also pointed out that some components of the mother
liquor such as isopropanol may enter the oil phase. This was anticipated by
Hope in the original papers describing the use of oil and Henry wrote "...
things like isopropanol can partition into the oil. I may be wrong but I
think that Hope would saturate the Paratone with the mother liquor by
shaking them together and then letting the phases separate."
5. Henry Bellamy also pointed out that "You need to be very careful about
immersion oil, it is selected for refractive index and can contain all kinds
of stuff. Back in the 80's Ivan Rayment was sealing Linbro plates with
immersion oil and he could only get crystals after he switched to another
sealant. So if things don't work the immersion oil is a good first choice
for something to change." If anyone would like to try the
oil/cryo-protectant technique I would recommend using paraffin oil as this
avoids the above problem, is cheap and worked the best out of a range of
oils I tested including the 4 Hampton immersion oils and ParatoneN. I used
LABCHEM brand bulk paraffin by AJAX chemicals but any lab-grade paraffin
6. Peter Moody had experienced problems when using a 50:50 Paratone/paraffin
mix with the oil being used as the sole cryo-protectant. The oil mix took up
some of the aqueous phase during xtal mounting and become cloudy when
cryo-cooled to 100K.
7. Ho-Leung Ng pointed out that a convenient bath of liquid N2 can save
rushing to the cryo-stream. However this still requires the use of
cryo-transfer and the oil/cryo combo is much quicker when screening a lot of
xtals and does not have the risk of damaging a xtal via momentary warming.
Thanks to everyone for the input and advice and if anyone else would like
more information please send me an Email and I will send a detailed
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