Guide to the Art Robbins Phoenix Crystallization Robot at Princeton

The Art Robbins Phoenix is an efficient crystallization robot that is quite effective at setting up small drop volume screening trays. This document is designed to highlight the actual operation of our setup at Princeton - the robot falls under the umbrella of the Macromolecular X-ray Crystallography core facility. There is a PDF manual for the machine on the Desktop of the control PC that is more detailed than this document. The content overlaps this one, especially in the programming segment.

This is an older picture when we had it set up on a lab bench, but the current layout is essentially the same:

The robot itself is the blue box in the middle, but the robot requires a small flotilla of other devices to drive it.

• Computer (far left) to control operations
• Vacuum/compressed air source to dispense solutions (black box to left of robot)
• Liquid pumps to dispense wash solutions (to right of robot)

Safety

Operations

Overview

Here's how you position a crystallization tray. Note the metal pins that hold the tray in location, and there are springs at top and right that hold the tray in place. Make sure it's seated well and the A1 drop is front left (lower left in this picture). This tray is installed at position 2 on the upper deck. The only way the robot knows which media/format is in which location is by how the layout is defined in the software protocol. It's important that tray and block locations match the protocol, so use a standard layout and stick to it. Additionally things like crystallization trays come in multiple formats. We use the "Intelli-Plate 2-well Deep Reservoir" trays exclusively to simplify this. Deep well blocks and the protein chill blocks are also a standard format.

Here's a deep well block containing a screen - these are manually loaded. Remove the foil seal before use and place the block in one of the lower deck locations - usually #8. Reseal with foil after you're finished with them. Be sure to remove the residual glue from the seal on the top of the block, since this can wrap itself around the needles. It can also make the syringe guide plate sticky, which can pick up your tray and trash the 96-syringe head.

The robot itself has two dispensing heads:

The one on the left is a 96-syringe block that dispenses 96 wells of solutions at one time and has a guard plate to keep the needles aligned. These needles are flexible, and you may see them bend slightly when dispensing solution into the 96-well crystallization tray. This is normal. However if a needle bends beyond 90 degrees then it is getting stuck in the metal guide plate and you should halt operations until this is fixed. Sticking needles are due to one of two things: failure to wash the head, failure to just the "Above Bottom" parameter in the Aspirate step of the protocol to match the liquid level in your block.

The one on the right is a single nano-needle used to dispense protein droplets. This needle cannot reach the lowest level. It sprays protein in the crystallization plate via ink-jet type technology, so it never drops down into the plate itself. This needle is both stiff and fragile, and once bent has to be replaced at significant cost ($250). We go to quite some lengths to protect this needle and keep it clean, since it is vital to correct operation.

And, finally, this is the wash station with the four pumps that drives the wash cycles for the nano-needle and 96-syringe head. The large conical flash is filled with water with 0.02% azide. There's a 100x (2%) azide stock kept nearby for your use. Don't let the flask dry out - fill it with water+azide when low. Doing a lot of trays with the associated washes can consume quite a lot of wash water - check regularly.

Start Up

• Turn on system using the Tripp-Lite power strip sitting between the computer and the robot
  
• If the PC is not already on, turn it on (and leave it on)
• Launch the Phoenix application via the icon on the Desktop
• Use the Connect button at top right of the program menu bar to connect to the hardware
• Watch until initialization is complete (status bar at bottom of screen will be blank), Connect icon will now show Disconnect
  (larger version)
• Put your empty crystal tray and the deep well block for your screen in known locations on the robot. Typically top right (location 3) is for the protein yellow block, middle top (location 2) is for the tray and the deep well block on the lower level (location 8).
• Program the run you want to do

Shut Down

• Fill out the log book entry
• Run the 0.1M NaOH/2% Detergent wash protocol
• Disconnect the software from the device ("Disconnect" button)
• Close the software
• Turn off the power block (Tripp-Lite)
• Leave the computer running

Opening and modifying existing protocols

• Click on the Open Protocol icon at top left. In the Protocol Manager window click on the Creator header to sort by creator name, which is nearly always the most effective way to find your protocol. Select, then click on Open Protocol.
  
• You're welcome to use other people's protocols, but make sure you fully understand what they do before using them. You can make a version for yourself using the File>Save As option, but please change both the protocol name and creator name otherwise you'll overwrite somebody else's protocol.
• Deck Layout. At the top of the protocol under "Protocol Start" the table at the top right shows you the layout as you've defined it for the protocol. You can drag elements from the lower right panel into the 9 deck positions. Unless you have a really good reason to do otherwise, put the crystallization tray in location #2, the chill block in location #3, the screen in position #8 and the Wash Block for the 96-syringe block in #7. You specify which of the locations each of the subsequent protocol steps refers to, so get this right and keep it predictable, otherwise you'll dispense your protein into an empty space or a wash station. The software relies on you to set this correctly so putting a chill block in a position where the robot expects to find a crystal tray will mean that you trash the nano needle. We will not be amused. The tray we use is the "Intelli-Plate 2-well Deep Reservoir". Quite the name, that.
  
• The core of any protocol is the ASPIRATE and DISPENSE steps. Nano-aspirate and Nano-dispense pull up and spray protein using the nano-needle. Aspirate and Dispense pull up and pipette screen solution using the 96-syringe head. You adjust the volume of these aspirate and dispense steps to change the drop volume and relative ratio. There two aspirate (protein; precipitant) and three dispense steps (precipitant in well; precipitant in drop; protein in drop), so a total of 5 steps. You can make more elaborate protocols (e.g. to dilute down the precipitant solution) but these five steps exist in all standard protocols in one form or the other. Preceding and subsequent steps are mostly concerned with textual reminders ("Message" steps) and washing of the needles.
• Aspirate step. Pull up 65 ul of screen solution from the deep well block in location #8, for every location in the block. It's OK for some block locations to be empty, since there's no way to have it aspirate from a subset of the block. You must have removed the foil from the screen and cleaned up the surface to remove residual glue. CHANGE THE "ABOVE BOTTOM (MM)" VALUE TO REFLECT THE HEIGHT OF THE SOLUTION IN YOUR SCREEN BLOCK. AVOID COATING THE ENTIRE NEEDLE WITH VISCOUS PEG SOLUTIONS SINCE THIS MAKES THEM STICK IN THE GUIDE PLATE. The needle only needs to be a 5mm below the surface of the solution to aspirate correctly. We always aspirate a few ul more than we are going to dispense, to allow for pipetting error, especially for PEG solutions. Note that you specify the volume for each well, not the volume for all the wells combined (i.e. 65 ul not 6500 ul).
  
• Nano Aspirate step. Pull up 80 ul of protein from the protein chill block in location #3, with the tube located at A1. ALWAYS REMOVE THE CAPS FROM TUBES SITTING IN THE PROTEIN CHILL BLOCK - WE'VE HAD INSTANCES OF PEOPLE FORGETTING TO UNCAP THE TUBE AND DESTROYING THE NANO-NEEDLE. Always apsirate a few more ul than you'll need to dispense into the tray, to allow for imprecision in the nano-needle. If material is precious, you can add a step to the protocol to have it dispense the residual back into the protein tube. Note that since the nano-needle dispenses into each drop, this is the total amount of protein needed for the tray, not just for one drop. (Nano-aspirate is different to Aspirate in this manner).
  
• Dispense to well. Puts 60 ul of solution from the 96-syringe block into the well (highlighted in purple) in the tray in location #2. Location (crystal plate) is selected at top right, and the sublocation within the tray is selected at center right.
  
• Dispense to drop. Puts 0.7 ul (typically more like 0.3 ul) into the larger drop location - highlighted in purple - of the tray. For the 96-syringe head you can't pipette into a subset of the tray - it's an all-or-nothing thing and all the pistons move in synchrony. For smaller drop volumes you might have to lower the "Distance Above Bottom (mm)" to nearly zero for the drop to make contact with the plate. The syringe needles are flexible, so they can accept a value of zero.
  
• Nano Dispense step. As shown here, this dispenses 0.7 ul into each of the 96-drops of the tray. Normally we'd do more like 0.3 ul. The tray is selected as sitting in position #2, the locations as "A1 to H12" i.e. the entire tray, and the destination is the larger of the drop depressions. You should have aspirated at least 100x the individual drop volume for this step to work (i.e. > 70 ul in this case).
  
• At this point the protocol presents the tray to you so that you can seal and label it. It's a very good idea to check that the drops look as you expect and that dispensing protein and precipitant solution appears visually reasonable. The protocol then goes on to wash both nano-needle and the 96-syringe block with water.
• The final procedure is the wash protocol you can find a NaOH/Detergent wash protocol under many user's protocol lists and Save As... your own version. It requires you to put 2% Micro90 detergent as 2 ul detergent in ~100ul water in position A1 and 200 ul of 0.1M NaOH in positions B1 and C1. There's usually stock 0.1M NaOH adjacent to the robot. The detergent lives in a 10ml to the left of the robot. Then run this protocol which extensively washes both the nano-needle and the 96-syringe block. Remember to keep enough water in the large flask to complete all the washes (add azide to the wash water to 0.02% from the 100x stock).
  
  You must perform the NaOH/detergent wash of the robot at the end of every run (not every tray) or when leaving the robot idle for more than one hour . This applies even if you are making a whole series of trays, because otherwise the needles can get clogged. You should wash the nano-needle and 96-syringe head with water during each protocol, also, otherwise you're likely to get solution contamination.

Switching Media (Tray) Types

The list of media known to the robot is visible in the table at lower right when you click on the Protocol Start header. If you want to use 96-3 three-drop plates instead of 96-2 original deep-wells, or switch to 96-2 shallow well plates you're going to have to modify your protocol. Best to make a copy of it first with a different name using File>Save As.

• Click on Protocol Start at top of protocol to bring up layout view
• Drag the new plate type from the table at lower right onto the layout - nearly always position 2
• Your protocol will show errors adjacent to all the steps that Dispense into the tray, since the definition is no longer valid
• Click on each step with a "No Matching Plate on Deck" error, click on the plate location in the layout
• The error will switch to "No Layer Defined" and click on the tray layout to define the dispense destination (well, drop1, drop2 etc.)
• Save protocol, run dummy test to check 96-syringe dispense and protein dispense into the new tray

Programming the run from scratch

First step is to load the "deck" panel with the media you are using. This tells the program what plates and deep well blocks are in use and where they are. You'll also need to specify the location of the protein chill block (usually in position 3) and the 96-syringe wash block (usually in position 7).

Click on Protocol Start to bring up the media table and the layout. Click-drag media from the table to the layout. Each location on the three metal decks of the robot are numbered from left to right: 1,2,3 are on the top deck; 4,5,6 are on the middle stage; 7,8,9 are on the bottom deck. The protein delivery needle can reach locations 1-6 but not 7-9 so there's no point putting either the tray or your protein there. Always put deep well blocks and trays such at the A1 location is at the front left . There are spring-loaded locations for each tray. Make sure that your tray is sitting square in the locations since the robot uses basic geometry to go to where it expects tray wells (etc) to be.

Then make your protocol - which is specific to the layout you just defined - by Click-Dragging specific steps (Aspirate, Nano-Dispense etc) onto the protocol.

The robot has two solution delivery systems. The Nano needle is a single needle that delivers the protein using a method like that of an inkjet printer. The program refers to modules using this needle with the prefix Nano (Nano Dispense, Nano Aspirate). Any other module (Dispense, Aspirate, Mix) is using the head at the left of the machine that contains 96 needles that deliver solution at the same time.

1. Wash (washes to 96-syringe block)
   • 3x repetition
   • Volume 100 µl
   • Distance above bottom: 2 mm
   • Liquid class: Aspirate water
   • Fill time: 9
   • Empty time: 12
2. NanoWash (washes the protein nano-needle)
   • Volume 200 µl
   • Check to see that needle aspiration blows bubbles in wash solution during washing
3. Aspirate (loads screen solutions into the 96-well syringes)
   • Identify screen block location (arranged with A1 on front left) on the decks.
   • 62 µl (allows 60 µl well + 2 µl for drop) - this volume is PER well+drop
   • Air gap: 0
   • Pre-dispense: 0
   • Distance above bottom: 2 mm (for less than half-empty blocks) or 15 mm (for more than half-full)
   • Liquid class: Aspirate screen
4. Dispense (deliver solutions to well from 96-syringe block)
   • Volume delivery: 60 µl
   • Identify tray location on deck, and WELL location w/in tray
   • Distance above bottom: 1 mm
   • Tip touch: None
   • Liquid class: Dispense screen
5. Dispense (deliver solutions to drop from 96-syringe block)
   • Volume delivery: 0.2 µl
   • Identify tray location on deck (drop A1 on front left) and DROP location w/in tray
   • Distance above bottom: 0.1mm
   • Tip touch: None
   • Liquid class: Dispense screen
6. NanoAspirate (sucks protein up into nano-needle)
   • Define location of protein chill block and protein well (usually A1, front left)
   • Volume for a 96-well tray of 0.2 µl drops would be 96*0.2+10 i.e. 30 µl using 10 µl as dead volume
   • This volume is for ALL the drops, dispensed via inkjet-like mechanism.
   • VOLUME DEPENDS ON: what you've specified as drop volume in #5 Dispense (so for 0.2 µl the total is 30 µl)
   • Make sure at least 20 µl more than this in the protein vial
   • Distance from bottom: 0.5 mm
   • Liquid class: NanoAspirate
   • Predispense 2 µl x2
7. NanoDispense (dispenses protein to drop)
   • Define location of tray (as with drop/well Dispense step)
   • Select drop position (as with drop Dispense step)
   • Drop volume: 0.2 µl
   • Location: Fill All Plate
   • Class NanoDispense
   • Speed 25%
8. Exchange
   • Allows you to remove and seal plate, and recover protein solution as necessary
   • The operations freeze until you press return
9. Wash
   • parameters as step 1
10. NanoWash (wash the nano-needle again)
    • volume 30 µl - use the same volume as the NanoAspirate step
11. NanoWash (wash the nano-needle again)
    • volume 60 µl - use twice the volume of the previous step
12. NanoWash (wash the nano-needle yet again)
    • volume 200 µl
13. NanoDispense
    • desination B1 of protein chill block in which you should have a small tube
    • volume 1000 µl with the PURGE button clicked
14. Wash
    • use the same parameters as step 1, the 96-well syringe washing step
15. Wash
    • use the same parameters as step 1, the 96-well syringe washing step

Phil Jeffrey, first version: March 2010, most recent update: September 2016.