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Thanks to all who responded!
Here is the compiled 'tricks of the trade' for microbatch crystal mounting.
Rebecca
ORIGINAL POST
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.
SUMMARY OF RESPONSES
Sorry if this sounds a crazy suggestion, but sometimes the simplest things
work. Did you try to mount the crystal directly on a loop and see if it
diffracts. The oil can be cryoprotectant.
The setup would be:
1- fish the crystal with a loop
2-do not care if it cames across the oil layer and it retains the oil.
3-mount in the cryostream
4-shoot X-rays and see if you have diffraction
******************
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.
******************
In one case I had crystals grown under paraffin oil in microbatch, which
required 25% glycerol for cryoprotection. However, when I transferred them
directly to the base condition + 25% glycerol, they would invariably break
up. So I ended up picking up a crystal with a loop, pulling it through the
oil, and putting it on a coverslip. Then I removed the surrounding liquid
and quickly replaced it with 4 microliters of base solution + 5% glycerol,
observing the process under the microscope. The crystal seemed to hold up
fine, and I then progressively replaced the solution with base condition +
10%, 15%, 20% and finally 25% glycerol. The resulting crystal froze nicely,
and provided a 2A dataset. Any oil that was still around the crystal from the
first transfer was probably completely removed by the cryo/washing steps, as
other people have already described.
******************
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
<https://email.brown.edu/exchweb/bin/redir.asp?URL=http://www.bioc.aecom.yu.edu/labs/blanlab/VETTING/PROTOCOLS/vapdiffunderoil.html>
if you want to check out our VDUO setup.
******************
We do the same, I prepare 2-3 drops of cryoprotectant on a microscope slide,
draw the crystal through the oil and "wash" in successive cryo drops until I
see no more oil coming off. Paraffin oil itself is mot a good cryo
protectant, if you want to use paratone oil you can use the same procedure,
or first get rid of the paraffin oil in artificial motherliquor drops, then
tranfer to paratone.
******************
I have very tough time mounting crystals from
microbatch. Essentially, I was adding the
cryoprotectant to the drop and then transferring
crystals to another drop of cryoprotectant. Yet, I
lost many crystals before successful mounting. At
times, I tried to remove the oil layer and then add
cryoprotectant, but it was always difficult.
******************
I harvested exclusively from microbatch under oil for a little over half a year
due to a crystal form that grew in 20%+ volatile alcohol (hanging drop tended to
swirl about something fierce when unsealed). I found that the main problem is
getting the crystal up through the oil, as paraffin oil tended to knock crystals
out of loops when pulling out of the aqueous layer. Al's oil is a little easier,
but still not a cake walk.
However, residual oil on the surface of the drop/loop didn't significantly
affect diffraction. It was a little harder to see the crystals in the loop with
blobs of oil getting in the way, but not impossible
A few schemes that worked well for me:
1) Step-wise supplement the drop with small volumes of mother liquor +
over-concentrated cryo. For example, if starting with a 2ul drop, add 0.5ul at a
time of mother liquor + 30-40% glycerol. Once you've done this 4-6 times, you'll
have (relatively) gently moved into 20% glycerol while staying under oil.
Different crystal forms may demand different steps/concentrations of the cryo
stock.
or...
2) Use a pipete to remove all but a thin layer of oil from the top of the drop
and then increase the drop volume to 4-6ul with mother liquor. This leaves a
thin film of oil to slow down evaporation, but isn't quite so hard to pull
crystals through. If you aren't in a volatile condition, you could just remove
all of the oil by flooding the drop this way . Then transfer to a separate well
with cryo (also covered with a slick of oil, if the condition is volatile).
3) As for crystal/loop handling, the hanging drop method of setting the loop
parallel to the surface of the drop and then pulling up is awful for microbatch,
as this causes the oil to shove the crystal out of the loop almost every time. I
found that 45 degrees to nearly perpendicular to the surface of the drop tended
to work best.
******************
A nice way to use cryo is to add it under the oil into the drop let it sit for
the time you need and just fish it with the oil then the crystals will be
already protected. It worked for me and others in the lab and it also enlarge
the volume of the drop so you can easily fish.
******************
I don't typically use the oil as a cryoprotectant.
First I remove *most* of the oil from the plate, and then remove the
bulk of the oil from the well where I'll be working. Next, I use a
piece of clay to hold the plate at an angle, which makes it easier to
get at the well. Then, I either
(a) fish the crystal directly from the well (through the remaining oil)
and transfer it to a small drop of cryoprotectant on a cover slip; or
(b) I use a pipet to transfer the drop to a cover slip and then work
from there (in the latter case, you'll bring some oil along, which will
coat the drop after you deposit it on the cover slip, which helps
prevent evaporation and allows you to perform the subsequent steps at a
leisurely rate).
******************
We pour off the oil and then add a harvest solution (~10-20 ul to
each terasaki plate well) with ~2% more PEG than that in the droplet
(remember that the drop concentrates with time - sometimes fine
tuning of the harvest solution PEG concentration is required but
generally 2% works.). This keeps the crystals stable and displaces
any oil remaining at the top of the droplet. Sometimes there is a
skin on top of the droplet. This is removed by a capillary or
acupuncture needle. Then we remove the crystals as per sitting
drop. We have tried to cryo-protect the crystals by just pulling
them up through the oil but this has not worked for our crystals. It
may be possible to add a cryoprotectant to the harvest solution but
we haven't tried it.
******************
Comments
1. Most people simply leave the oil, and loop the crystal out through
the oil
2. The crystal must fit the loop well, or the surface tension will drag
it off the loop
3. People who are used to working with oil claim it is easier than
sitting or hanging drop because the oil prevents evaporation. Therefore
you don't have to work fast.
4. A loop with a bent handle can be helpful, because you have to
harvest crystals from above.
5. Tip from Jeroen Mesters: if you want to use the oil as a
cryoprotectant, you must DRY the oil first. Use ordinary paraffin oil,
and put 1 ml aliquots into Epi-tubes. Dry tubes e.g. in a SpeedVac
overnight. Freeze tubes, and use for a day then discard. To use as a
cryoprotectant, place a drop of dry oil on a glass slide. Drag the loop
with the crystal through the oil along the surface of the glass. Small
drops of mother liquor will stick to the slide. Remove as much mother
liquor from around the crystal as possible, and freeze in the normal
way. (Have I got that right Jeroen?)
6. Use Vapor Batch plates rather than e.g. Nunc HLA trays, because they
don't leak oil on the bench! See
http://www.douglas.co.uk/products.htm#Vapor%20Batch%20Plates
<https://email.brown.edu/exchweb/bin/redir.asp?URL=http://www.douglas.co.uk/products.htm%23Vapor%2520Batch%2520Plates>
******************
I've mounted many crystals from microbatch and sitting drop plates and
haven't found it diffucult to mount xtals from either ! (well, of
course a bit difficult when i first started mounting xtals !!)
typically I try to remove the oil by dragging the xtals gently in the
cryoprotectant drop until i see the oil layer is removed (at much as i
can judge visually !) it's a tricky job removing the oil completely
depending on the cryo (eg. paratone !!!!) so great attention is needed
in doing this part ! and I tranfer the xtals to a fesh drop of cryo
before mounting.
******************
1) Try to change to hanging drop vapor diffusion, by halving the percipitant.
2) Flood the microbatch drop with the first stabalizing / cryo
solultion, then remove the oil layer
******************
We usually dip the loop straight through the oil drop and try to 'fish' out the
crystal. We then transfer it to a fresh drop of cryosolution and scoop it back
out before mounting it in into the beam. This however isn't as easy as it
sounds and takes a little practice. We do sometimes remove the oil drop
(partially) to make it a little easier. Further more if we use oil as a
cryoprotectant, we would transfer it to a paratone (not paraffin oil) drop
before mounting the crystal. I have never heard anything about using the
paraffin oil on top of the drop as a cryo protectant. However I found out that
using paratone as a cryoprotectant is also very difficult, and as for al the
described techniques practice makes perfect.
