Microlytic is firmly committed to ensuring that our customers have access to the newest and most optimal protocols for Crystal Former on various robotic platforms. Our in-house experts are available to assist you in most steps of Crystal Former programming and to serve as liaisons with the respective robotics manufacturers to assist in custom programming and/or integration on new robot units. If you unit is not listed below, please contact Microlytic support directly for information on how to proceed.
Mosquito plate and protocol files are available from Microlytic by contacting firstname.lastname@example.org. Please specify the number of decks on your Mosquito unit to get the correct protocol file. Your software version must be 2.8.58 or newer for 2-deck (Crystallography deck) and 5-deck units, and 3.4.1 or newer for LCP 4-deck units. Older software will crash when attempting to open the plate file
TTP strongly recommends that your Mosquito is calibrated prior to setting up Crystal Formers, as the Crystal Former plates have much smaller margin of error with tip positioning than vapor diffusion plates. To keep plate setups running smoothly users are also advised to calibrate their unit regularly, as per manufacturer specifications.
If you are creating your own protocol files, we recommend incorporating the following items:
Dispensing speed should be to be set to 1 mm/sec to reduce drop beading at the channel inlet
A pause needs to be inserted after protein dispensing to give protein sufficient time to fill channels completely. This is particularly important for viscous protein samples.
Standard plate definition files work well on units that are properly maintained. Some units may require needle position adjustment on x, y, and z axis in the “Plate” tab of the protocol file. Fine tuning is best accomplished by activating “Enable single step mode” function in the “Options” menu once the run begins. This allows for observing the position of the needles in the inlets.
Tips should not flex or push down on the backing of the plate during a dispense, which can cause the backing to leak/detach. If this happens, increase the head height. The standard head height is 6.35 mm above the base of the plate
When the protein sample is set up, it is prudent to examine whether the channels of the Crystal Former had sufficient time to fill with protein during the “Pause” step. If any protein drop beading is observed at the inlets (due to high viscosity of protein sample or sub-optimal needle positioning), a strong tap of the plate on the bench is sufficient to shake the droplets down towards the channel inlets. Another tapping step after precipitant addition will ensure good liquid contact.
The minimum volumes are 300 nL per well for protein and 500 nL for precipitant. If the protein volume appears to be insufficient to fill the channels, calibrate the unit and make sure the tips are loaded into the clamp correctly with no splaying. These minimum volumes may need to be increased if incomplete channel filling occurs due to higher viscosity of the protein sample or suboptimal needle positioning.
Plates and protocol files are available from Art Robbins technical support at email@example.com
It's most important that you have the 96 needle head properly calibrated to all of the teach points where you intend to use the Crystal Formers. Use paper clips to lift up the metal plate to expose the needles for calibration. Using a flashlight helps to see the alignment of the A1 needle over the teach point
Make sure your nano needle is properly calibrated to all of the teach points where you intend to use the crystal formers. This isn't as sensitive as the 96 needle head, but it should still be checked.
Use pre-dispense after aspirating both protein and precipitant to get rid of any air bubbles
We recommend inserting a "pause" step after your protein dispense in the protocol - it's best to make it an indefinite pause requiring a prompt. You can also use the “purge” function for the nano head to move it out of the way to access the Crystal Former plate easier. At the pause you should remove the Crystal Former from the deck and give it a sharp rap on the bench top to ensure all channels are filled (this shakes off any droplets clinging to the inlet walls to make contact with the channel opening). Replace the plate and then proceed as normal.
The sample protocol should look something like this:
Wash 96-syringe head
Wash nano head
Aspirate screen into 96-head (say, 5 uL)
Pre-dispense screen back into the plate (3 x 1 uL)
Pre-dispense protein (3 x 0.3 uL)
Dispense the protein into Crystal Former (300 nL/channel minimum)
Purge the remaining protein from nano head back into the protein tube (to move the head out of the way)
During the pause take the Crystal Former out and tap it very firmly on the bench a few times
Place CF back onto the stage and resume the protocol
Dispense 500 nL of precipitant, use tip touch to all 4 sides of the inlet to make the drops stay in the plate
During the head washes take CF plate out and tap it on the bench a few more times to shake down the precipitant droplets
If you ensure that you have done all of the above steps, you should have no issues using the Crystal Formers on your robot.
Plate and protocol files are available from Formulatrix technical support
Robot requires no calibration or adjustments prior to setup
Tip wash is recommended between each dispensing step
Ensure that protein and precipitant are always dispensed at 1:1 ratio on this robot. The minimum recommended volume is 300 nL
When working with minimum recommended volumes, the liquid class of solutions may need to be adjusted to slow down dispense for more viscous solutions
After the protein dispense, pause the experiment, take the plate out at tap it couple if time on the bench to ensure that all droplets have made contact with the channel opening.
Repeat the tapping step after the precipitant dispensing
Files are included in the software For Oryx 4 and 8
Technical support may be obtained by contacting firstname.lastname@example.org
Protein: minimum of 250 nL per channel,
Precipitant: 300 nL - 500 nL (manual dispense)
Files are available from LabCyte technical support. Please see the website for region-specific support e-mail address: http://www.labcyte.com/support
250nL protein+ 250nL precipitant for CF-HT2
150nL protein+ 150nL precipitant for CF-O
Support for Crystal Former imaging is available directly from Formulatrix. Requests for Crystal Former automation should be submitted to Formulatrix support, specifying the protocol for 4 images taken per channel. This imaging strategy will ensure sufficient magnification of the channel contents to ensure that more microcrystalline successes are not missed. Imaging protocols for single images per channel are also available, though the user is recommended to include some manual plate inspection with this protocol.
Support for Crystal Former imaging is available directly from Rigaku for Minstrel DT and HT units that are not considered legacy units. For information and recommendations on programming legacy Minstrel units, please contact Microlytic support.
The Minstrel DT system defines the channel within the context of a 3 x 3 imaging grid, which is due to software limitations on the imager. The result is that two of the images are not informative, as they capture only the middle region of the well position that does not contain any part of the mcirochannel. For the HT units, the sofware is devoid of this restriction and imaging is carried out sequentially along the channel.
All Crystal Former formats are compatible for imaging and diffraction testing on the PX scanner. Please refer to our whitepaper in the documetation section for additional information All support inquiries should be directed to Agilent for these units.