Tecan crystallisation robot user guide The TECAN crystallisation robot in RRB S9 provides an automated facility for setting up crystallisation trays. This user guide is meant as online support for trained users.

1. Creating scripts to dispense trays using the Tecan robot Traymaker is a javascript applet that allows a user to define a crystallisation screen in terms of solutions, concentrations, and gradients, in a way that can be archived, analysed, and exported as the sort of simple pipetting script that drives the Tecan robot (i.e. aspirate and dispense commands).

Logic The logic underlying Traymaker is as follows. A crystallographic screen employs one or more components. These components are the stock solutions from which the screen will be prepared. Each component will be pipetted into the crystallisation tray in one or more patterns. Each pattern transfers the stock solution in some sort of gradient from the stock solution tubes, and into a block of wells in the crystallisation tray. Specifying the components, their stock concentrations, and where you will put the stock bottles on the robots table is done using a nice™ gui. Similarly, specifying the patterns, the block of wells that they cover, and the gradient to use is done interactively. Don’t panic. Having a go (which you can do by following <a href=”this link “ http://biop.ox.ac.uk/www/noble/Traymaker.html”> ) will quickly show you that it’s easy to generate both extremely exotic and extremely simple pipetting scripts.

To create a Traymaker Script

1. Starting traymaker To run the Traymaker program you will be in one of two scenarios a) At your desk: Navigate your web-browser to http://biop.ox.ac.uk/www/noble/Traymaker.html When you have created a script, you should save it in Traymaker’s native (.xml) format, and export it to a Tecan script (.gwl) file. These can be burnt to CD or stored on a USB stick for transfer to the PC that controls the Tecan robot. That PC is connected to the internet, so can in principle navigate to file servers or other places you might store your data. b) At the tecan robot. Double click the Traymaker icon on the desktop.

2. Creating or opening a script. Under the 'File' menu select either 'new' or 'open' (to edit a pre-existing file). N.B. The menubar that allows you to read pre-made scripts into the applet, save scripts in Traymaker’s native format, and export a corresponding Tecan script will show up at the top of the traymaker page in the browser, not at the top of your monitor (like Mac applications), or at the top of your window (like linux or windows applications).

This will create a new window area: first provide a tray name (used as the default file name when subsequently saving the file in either .xml or .gwl formats). You are provided with two options to modify either 'wells' or 'droplets'.

3. Including a new component Select ‘component add'. This will activate the RHS of the screen that shows a schematic layout of the Tecan worktable:

You can (and should) specify some things about each component: namely a) Name (since you can print Traymaker output, this is a very efficient way of keeping records about what is where in your trays) b) Concentration and units of your stock solution. c) Position of your stock solution tubes on the worktable. By default the first four tube positions on the LHS 15 ml Falcon tube rack of the table are selected (red dots, see Figure above), but these can be changed by clicking and dragging the mouse over as many tube positions as required. hints i) as the Tecan has 8 tips, spreading each stock solution among 8 tubes will speed up the tray making as all 8 tips will be used simultaneously to dispense the solution. Using 4 tubes for each stock solution is a good compromise. ii) If you have 96 different stock bottles (e.g. tubes of Hampton screen reagents), these can be handled by selecting all 96 positions. Stock solution Column 1, rows a to h will be pipetted into the crystallisation tray column 1 row a-h. Stock solution column 1, rows i to p will be pippetted into column 2 rows a-h etc). d) PLEASE DO NOT CHANGE THE 'pipettes like....' TEXT.

4. Editing the default pattern

You can vary the following aspects of the pattern: a) Cells into which the component should be dispensed. To modify, click and drag a box over the wells you wish to select. N.B. By default, the destination for pipetting is the top crystallisation tray position (top right of the worktable). To change this, select 'preferences' from the 'edit' menu and then select a new destination from the menu that appears when you click the 'destination tray' button. b) Gradient type (may vary from top-to-bottom, left-to-right, or continuously from the top left corner of your pattern to the bottom right) c) Concentration increment (may vary linearly or exponentially…if you select exponentially, remember not to start with a volume of zero!). d) Start and end conditions. This can be done by providing (i) start and end % of stock; (ii) start and end molarity (or whatever unit you have specified your stock concentration in above); or (iii) start and end volumes (in microlitres) Hitting return after entering values by any of these mechanisms will cause the other mechanisms to auto-calculate.

5. Adding further patterns If you wish to dispense the same component in a different pattern (e.g if you are doing repeat operations on the left and right hand sides of your tray), then click “Pattern Add” on the left hand side of the traymaker window

6. Adding further components To add more components, repeat steps 3-5.

7. Modifying existing patterns and components Returning to the top of the page you will find you have created a folder named after your stock component 1 solution and within this 'pattern 1' where the pattern details are stored. This can be edited at any time by clicking on either to select. To delete the component or the pattern, first select and then scroll down to the bottom of the page and select either 'delete component' or 'delete pattern'.

8. Saving and exporting To save the file in .xml format (can be read by traymaker), select 'save' from the 'file' menu and name the file with extension .xml. To export the file in .gwl format (can be read by the Gemini software that drives the tecan), select 'export' from the 'file' menu. At this point you will be asked if you want to top up the wells with water. (Well solutions are made up to 100 microlitres). Tecan export scripts must be given the file extension “.gwl” N.B. If you are editing a script on the PC by the Tecan, please save your files within your own folder within the 'MyDocuments' . NOTES The components will be dispensed by their order in the script, so consider adding buffer and salt solutions before more viscous PEG, or more volatile organic reagents. 2. Running scripts to dispense trays using the Tecan robot To login: To log on to the PC that operates the Tecan, use user name “Administrator” and the password “Tecanadmin”. The Gemini software username is Tecan and the password Tecan 1. Preparing the robot Before starting your run (i) Check that the system liquid container (see picture below) has enough liquid to last the duration of the run. On average, a run uses 1 litre of system liquid. If the system liquid container is empty, fill it up with 1% isopropanol in MilliQ and degas using Argon for about half an hour.

(ii) Next, please check that the waste liquid container is not full when starting your run. If the container is full, there is a risk of flooding the robot’s work surface. To empty it, you can use the sink next to the heavy atoms cupboard in S9.

(iii) Check that the MilliQ reservoir is full (the grey container of three closest to the front of the worktable). Depending on the tray to be dispensed, the robot will have to be configured with 15 ml Falcon tubes, 2 ml deep block(s) and/or 96-well crystallisation trays: 1. 15 ml Falcon tubes Check that all stock solutions are in their correct positions and carefully aligned so that the tubes won't collide with the tips. 2. 2ml deep blocks Check that the block(s) are in their correct positions on the worktable (position 1, 2 and/or 3) and that they are the correctly oriented (Lettering to the LHS, "A" in the top left hand corner) 3. The 96-well plate The robot is configured to accommodate MRC-LMB 96 square well plates. When putting your plate down on the plate rack, ensure that it is the right way up (row letters pointing towards the left, "A" to the top LHS). Gently press the black button on the upper right of the rack to accommodate the plate and release slowly to secure the plate in place. 2. To run a Traymaker script 1. Drop the Execute Traymakerscript.gem icon on the Gemini icon. 2. This will open a window within Gemini in which there are listed a number of worklists to execute a complete tray dispensing run that includes washing and cleaning the tips after a tray has been dispensed. DO NOT CHANGE ANY WORKLISTS EXCEPT WORKLIST 5. 3. Select worklist 5 and replace with your traymaker script. This can be done by double-clicking on the worklist and using the pop-up window to browse through files to select your file. DO NOT EDIT ANY OF THE OTHER OPTIONS WITHIN THE BROWSE WINDOW.

4. Once you have selected your file click 'OK' and your script will replace that of the previous run. 5. Before running the script, check that there are no errors by clicking on the 'blue tick button' to validate the run (see picture below). If there are no errors in the script, this window will pop up:

5. To run the script press the 'green triangle button' (see picture below).

nb. If you do experience difficulties, avoid pausing or aborting a script- instead switch off the robot at source. This will avoid any further damage and the robot can then easily be restarted. However, do seek assistance at this point. 3. Using Standard Scripts There are a number of standard scripts provided that will: 1. Dispense standard screens (in 10 ml tubes) to 2 ml deep well blocks (ImprintToDeepWell1.gwl, ImprintToDeepWell2.gwl, and ImprintToDeepWell3.gwl) 2. Make an imprint of a standard screen from a deep well block to a 96 well crystallisation plate (screen transfer deepwell1 to plate1.gwl) These scripts can be found in the folder 'Standard Transfers' within the 'My Documents' folder.

The file you will most frequently use is 'screen transfer deepwell1 to plate1.gwl'. The deep well 1 block position is the grey deep well block holder towards the back of the work table and the plate 1 holder to use is again the 96-well plate holder to the back of the work table. There are many variations of these dispensing steps that can be carried out readily using the Tecan (eg to make bespoke deep well blocks from stock solutions). If you wish to pipette more exotic patterns, please discuss with Martin Noble.