All systems (OryxNano, Oryx4 and Oryx8):
Sitting drop down to 100+100nl for screening
Up to 2+2µl drops for crystal harvesting and soaking
Contact dispensing allows "MMS" and regular microseeding
Only 10.0µl of protein for 96 wells (100+100nl)
- 2D grids
- Cross-Matrix (Combinatorial) experiments
- Additive Scatter Optimization
In addition - Oryx4 and Oryx8:
Hanging drops up to 8+8µl
Microbatch-under-oil down to 100+100nl
Oryx4 can be upgraded to Oryx8
Lipidic Cubic Phase (LCP) dispensing option
videos, and here for the advantages of the
All Oryx systems use multi-bore dispensing tips (microtips), which
have several independent channels to dispense small volumes. At the end
of e.g. a 3-bore tip there are three holes. Each channel dispenses a
different solution. The solutions do not mix in the tip - they mix in
the drop after they are dispensed. This means that there is no
dead-volume. Oryx8 has two chassis and can use 3, 4 and 7-bore microtips.
The microtip is on the left-hand arm.
• The microtip always touches the plate when liquids are dispensed.
This gives very reliable dispensing, especially when suspensions of e.g.
seed crystals are used, which makes the system ideal for
• Stock solutions do not come into contact with the motorized syringes, which are filled with degassed pure water - this avoids the need for
flushing out the syringes when the stock solutions are changed.
• The manual syringes on the front panel
are used to refill the motorized syringes, to remove air bubbles, and to
load stock solutions for optimization experiments.
• Large volumes, including oil for
microbatch experiments, are dispensed by the right-hand arm using the
large (2 ml) motorized syringe. A 1 ml disposable tip is used for this.
Screening for sitting drop is carried out using plates that are
prefilled with reservoir solutions. (Reservoirs can be filled manually
with a 12-channel pipette, or large-volume dispenser such as the
Liquidator 96 by Rainin). During each dispensing cycle, the tip is
first cleaned in the reservoir by
moving it horizontally through the solution. The tip then picks up e.g.
100 nl of solution from a clean part of the reservoir and transfers it
to the drop, dispensing protein simultaneously (together with seed stock
for microseeding experiments). The level of contamination has been
tested and is very low.
A very simple user-interface is used to design experiments, as shown
below. The correct plate can be selected from a database of all
well-known crystallization plates (if your favorite isn't there, let us
know!), the volumes
of each ingredient of a drop are entered in a simple form.
Individual wells can be selected or skipped by clicking on the plate
with a mouse. (The form shown below allows seed stocks and additives
to be added to drops, as
Exactly the same mechanism is used to dispense drops up to 2 + 2 µl for
crystal harvesting and soaking experiments, and down to 100 + 100 nl for
screening experiments. Smaller drops can be used but will not give
reliable results with some proteins. Evaporation is reduced by covering
the plates with a sliding shield (see below). For soaking experiments,
microseeding is highly recommended, see below. For information about
dispensing reservoirs for optimization experiments, see below.
Sliding Evaporation Shield
Since all Oryx systems use contact dispensing (the tip always touches
the plate) they give very reliable dispensing even when suspensions of
solid particles are used. This makes them ideal for microseeding
experiments. The technique of adding crystal
seed-stock to random
screens (rMMS) is a significant breakthrough in protein crystallization
that is very effective. For example, one industrial group
method to solve 38 out of 70 structures generated in a four year period,
finding particular success with antibody complexes. rMMS not only
produces more hits, it also typically generates better-diffracting
crystals – because crystals are more likely to grow in the metastable
zone of the protein’s phase diagram (see below).
Note also that in cases where only one or a few crystals are obtained in
screening experiments, the seed stock that can be made is very valuable
– often more valuable than the protein sample. It is therefore a great
advantage to be able to use the smallest possible sample of seed stock.
Using any robot from the Oryx range, seeding can be performed in a whole
96-well plate using only 1.5 µl of seed stock. This is particularly
helpful for membrane protein crystallization projects because membrane
protein crystals are often unstable and it is helpful to make seed
stocks without diluting the original mother liquor.
Videos about rMMS : (1)
Introduction and theory. (2)
rMMS experiments with an Oryx system.
Contact dispensing has another advantage: almost no protein remains in
the tip at the end of the experiment. Moreover, since only one
(multi-channel) tip is used, all of the protein for an experiment can be
placed in a single PCR tube, which also reduces waste. When they have
enough protein, most users set up 300 + 300 nl drops. For a 96-well
plate this requires only 29.4 µl of protein, i.e. only 0.6 µl is wasted.
If your pipette is accurate, there is no need to put more than the
specified amount into the tube!
Similarly if 10 nl of seed stock is added to each drop, only about 1.5
µl of seed stock is required for a whole 96-well crystallization plate.
(It is helpful to dispense around 5 µl of screen solution on top of the
All Oryx systems can carry out simple optimization experiments using three different approaches.
The standard screening software allows users to define simple 2-d grids
for sitting drop experiments. Using three or four ingredients, the user
selects the volumes to be dispensed in two corners of a rectangular
grid. The software interpolates linearly between those conditions. (The
user interface does not show the concentrations in intermediate wells,
but the volumes dispensed to each well are shown in a log.) The script
works well with plates with small reservoir volumes (e.g. the SwissCI
3-drop) because the maximum volume of any ingredient that can be
dispensed is 1620 µl. (A more sophisticated approach to optimization
that includes 2-d Grid experiments is available with the Oryx8 – see
Click here for a video
showing how to set up 2-d grids
with an Oryx
The systems' powerful combinatorial optimization” approach allows a
different additive or seed-stock to be added to each row. Each additive
is picked up from the corresponding PCR tube on the right of the table
(A1, A2 etc. on the diagram below). By arranging e.g. precipitants in
columns (P1, P2 etc.), different combinations of precipitants and
additives can be tested very quickly. This is useful for reshuffling the
ingredients of several hits, so that ingredients that are not helpful
can be eliminated quickly, and trends can be identified. For example,
certain ingredients may encourage the formation of crystals with certain
The combinatorial approach can also be to systematically identify the appropriate “dilution of a seed stock in a single experiment. We recommend using a highly concentrated seed stock
for routine rMMS screening, however this can result in showers of small crystals. It is often possible to optimize these conditions by diluting the seed stock to get around 5
crystals per drop (experiment with thermolysin shown above). For example, different concentrations of seed stock
could be placed in the PCR loading tubes shown 1 - 1E-6 dilution above. Four different conditions
could be placed into the four columns labeled P1 to P4 above.
This is a very effective way to get a really reliable supply of crystals
for data collection and soaking experiments.
Click here for a video showing
Scatter up to 5 additives (e.g. seed stocks) evenly distributed across a vapor diffusion plate. The vapor diffusion plate would typically be pre-prepared
with a 2D gradient of precipitant against salt or precipitant against pH. The robot will then distribute up to 5 additives in a
pattern across the 2D gradient. This tests the additives across a range of concentrations.
This experiment would normally be used for testing up to 5 dilutions of seed stock. It could also be used to test other additives or protein concentrations.
Features of the Oryx4 and Oryx8 systems only:
Oryx4 and Oryx8 can set up experiments with Linbro plates and cover slides, with up to 5 drops on each
cover slide. Volumes can range from 100+100 nl up to 8+8 µl (assuming 24 drops). You can also add one additive
to each drop with Oryx4, and up to five additives with Oryx8. This is very useful for leads that
are picked up in random microseeding experiments, where it may be necessary to add e.g. diluted seed stock to get crystals.
For example, in the experiment shown (below left), one drop has seed-stock, protein, reservoir solution; the second drop has no seed but
a little extra protein, while the third drop has no seed but a little extra reservoir solution.
You have to transfer the cover slides onto the Linbro tray by hand. After a few drops the experiment can be paused to allow the transfer.
Oryx8 can set up the reservoirs for optimization experiments, including 2-d grids and multivariate experiments (see below). The hanging
drop capability is not available for the OryxNano.
Additive experiments are a well-known and effective approach to
optimization. Simply by adding a set of potential ligands or another
screen to a hit condition, crystals can often be optimized. Since they
have larger Plate Loaders, the Oryx4 and Oryx8 systems can accommodate
an extra 96-well plate containing additives to the right of the target
plate. The tip picks up e.g. 100 nl samples from the additive plate and
dispenses them along with protein and reservoir solution to the drops.
For initial screening relatively large volumes of additive are often
used, for example 300 nl (protein) + 100 nl (reservoir) + 200 nl
(additive). For example, these volumes could be used for an initial
screen with the Silver Bullet screen by Hampton Research. For final
optimization, smaller volumes of additive are often used, for example
300 nl (protein) + 200 nl (reservoir) + 100 nl (additive).
Microbatch is a very simple approach to crystallization. Small samples
of protein (100 nl to 5 µl) are mixed with stock solutions in small
drops, and covered with oil to prevent evaporation. For screening
experiments it helps to use a 50:50 mixture of paraffin oil and silicone
oil. The silicone allows slow evaporation over about a month, which
gives a scanning effect across the phase diagram of the protein. For
optimization, pure paraffin oil can be used, which reduces evaporation
to a minimum. Studies have shown that microbatch finds as many or
slightly more hits that vapor diffusion, but the main advantage is that
(for reasons that may not be well-understood) certain proteins
crystallize much better in microbatch than other methods. Microbatch can
help to protect sensitive proteins such as membrane proteins and
anaerobically-produced proteins because it reduces the oxidation and
gives thinner skins on the surfaces of drops.
A full set of scripts for microbatch is available, including screening,
additive experiments, 2-grids and microseeding. Oil is put onto the drop by the 1
ml plastic tip just after it is dispensed.
Using the Combined MB-VD experiment
it is possible to dispense screening experiments for both microbatch-under-oil and vapor diffusion methods at the same time.
Using this procedure both methods and multiple sitting drop ratios can be dispensed in under 24 minutes. It is also possible to add seeds or additive to the drops.
Microbatch-under-oil 2-d grids
with an Oryx4 or 8.
Combined MB-VD screening
with an Oryx4 or 8.
The Oryx4 system can easily be upgraded to Oryx8 simply by adding a
second Chassis and changing the colors of the connectors. However, the
OryxNano cannot be upgraded easily because it has a smaller Plate
Loader. Contact the company for more details.
Lipidic Cubic Phase (LCP) dispensing is an option for
Oryx4 and Oryx8 (not available for Oryx Nano). The option is available for new systems
and can also be installed to recent compatible systems. An arm mounted positive displacement
syringe drive can dispense LCP volumes as small as 10 nl and up to 1 µl or larger. The shape of
the volume (dispensing technique) can be selected in the software. This allows the
choice of taller conical volumes or wider pancake shaped volumes (see image below). After the LCP
volume is dispensed the precipitant(s) and additives are immediately dispensed
covering the LCP volume and preventing evaporation or phase change of the LCP.
Oryx LCP machines are not able to dispense under oil
or vapour diffusion optimization experiments. This is because the LCP dispensing
arm is in place of the large volume dispensing arm. However it is possible to reconfigure the machine
between large volume dispensing and LCP dispensing modes in approximately 15 minutes.
Oryx4 and 8 LCP can dispense LCP to 22 mm or 18 mm hanging drop coverslips. This offers much better harvesting than a sandwich plate and better optics than sitting drop.
The Douglas Instruments manual LCP mixer is supplied with all Oryx LCP machines.
It allows simple preparation of LCP mixtures and because it is manually operated the user maintains kinaesthetic awareness of the sample during preparation.
The mixer is also available from Jena Bioscience.
Oryx4 LCP can dispense normal LCP screening and additive screening experiments. **We will soon add dedicated
experiment scripts for cross matrix optimization and
simple 2d grid optimization LCP experiments.
Video about LCP
with an Oryx LCP system.
** coming soon.
Click below for:
Oryx4 bench space
(Visio file - part of Microsoft Office.) Follow this link to see how much space is required on your lab bench
using the on-screen ruler. Click on the tabs at the bottom of the screen for
possible configurations. If no ruler is visible, click on the
between Oryx8, Oryx4 and OryxNano
Specification of MCC control unit
Getting started with Oryx4
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