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With the addition of the two Rock Imagers and Dragonfly to the X-ray facility, the ability to optimize your initial hits has never been easier. Optimization screens can be created two ways: create the screen in Rock Maker and export to Dragonfly; or create it in Dragonfly Designer and export it to Rock Maker. Most users will want to use the first method because it is simpler and you’re more likely to be optimizing from conditions you find in RockMaker.

RockMaker to Dragonfly

Create a new Experiment in RockMaker. Open the experiment with your hits and find the screen condition you wish to optimize (use control-click to select multiple wells), and then drag those well conditions to the new experiment. You can select “Grid Layer” or “Random Layer”. You can add conditions from several experiments by opening them up one at a time and adding them to your new experiment as above.

Grid Layer

When the conditions are added to the plate, you’ll find that all the conditions are identical to the well(s) from which they came. The number of grid layers you get on the plate will be the same as the number of wells you chose. To change how they vary, select each “Grid Layer” and in the right pane that opens up, edit each ingredient to vary along the row or column as you need.

Random Layer

If you want to create a screen made up of random components from several wells, select “In a Random Layer” after dragging well components to the new experiment. Depending upon the complexity and number of components, the entire plate may or may not be filled.

Export XML

When you are done, DO NOT USE THE EXPORT BUTTON ON THE TOP RIGHT OF THE PAGE. Instead, on the left sidebar, select the experiment (not the plate), right click and choose “Export to XML file…”. Print the barcode for your plate and you’re ready to go.

Import RockMaker-generated XML file into Dragonfly

In Dragonfly (NOT DF Designer), on the “Gradient” page, click the “IMPORT...” button. Make all necessary adjustments to plate type, well volume, and split dispensing of components that require it.

Run Dragonfly as usual.

Dragonfly Designer to RockMaker

Create a Screen in Dragonfly Designer

Design

When you open Dragonfly Designer (DFD), you’re presented with an empty canvas consisting of three things: Components; Region; and Gradient. Components are what your stock solutions consist of; Region is where  your stock solutions will go; and Gradient is how  (and if) your stock solutions will vary.

DFD1.jpg

Components

If you intend to put the plate you’re setting up on the Dragonfly (DF) into the Rock Imager and want to use Rock Maker (RM) to keep track of your well contents (hint: you do!), then TTP Labtech has made it easy for the components you enter in DFD to match the components listed in RM.

Click the “IMPORT” button next to Components. In the “INGREDIENT SEARCH” window, enter the name of the compound you’re adding. For example, if you type “PEG”, you’ll get a list of all the PEGs that are in the list. Select the one you want and then in the “INGREDIENT STOCKS” section, select the correct concentration of your stock solution. When you do, the “ADD SELECTED STOCK TO COMPONENTS” button becomes active. Click it to add it to the “SELECTED COMPONENTS FOR IMPORT” list. Repeat the above steps to find and add all your stock solutions that you’ll be adding. If you’re adding a stock solution that you made yourself, just add one of the commercial stocks that best matches your stock solution. When you have all of your stock solutions in the IMPORT list, click OK to transfer them to the Design window.

If you forgot something, you can click IMPORT again on the Design page to add more.

Regions

Once you have all your components entered, you can now arrange them on your plate. The default is for a 96-well plate, so when “Full Plate” is shown, this is what it refers to. The “resize” option is on by default, so you can make this region any size you like by clicking and dragging the corner of the highlighted area.

The plate we’re going to make will have two halves; both containing a PEG 1000 gradient; the left containing a NaCl gradient; and the right containing a CoCl2 gradient.

Gradients

Highlight the Region you want to add Components to and then click “ADD NEW” next to Gradients. This will add a line to the chart below and in the Component section, you can choose from the components you added earlier. Under Gradient, you can determine how the component’s concentration will vary. Horizontal varies along the row; Vertical varies down a column; Diagonal varies from corner to corner; and Constant keeps the concentration equal throughout. For each component whose concentration varies, you can enter a start and end value.

When done, it’s time to go to the Export window.

Export

The first thing to do is “CALCULATE OPTIMISATION” for your screen. Doing so copies the information you entered into the Design window and figures out based on the Well Volume (µL) entered how much of each component you’ll need. The Error List will show warnings such as the need to split components into two syringes or that the amount of one of your components is less than the Minimum Dispense Vol (µL).

When everything looks as you want it, you’re going to want to save THREE (3) files: one for DFD; one for DF; and one for RM. Click the “v” and select “save as…” to save a DFD file. Then, click the “EXPORT DRAGONFLY CSV” to save a DF csv file. Finally, from the dropdown list under the same button, find “EXPORT ROCKMAKER XML” to save a RM file.

Import DFD-generated XML File into RockMaker

On the Dragonfly computer, open Rock Maker and create a new experiment. On the Canvas tab, navigate to the top right of the window and select “Import” and then “Import XML screen”.

 

The next window that opens may seem a little confusing, but it is there to reconcile your stock solutions with the database of stocks in Rock Maker.

For each “conflict”, select the appropriate stock in RM until they’re all fixed. Choose the name of the ingredient (highlighted in red if in “conflict”). Under “Choose Action”, select “Use substitute ingredient” and then at the bottom right of the page, select the ingredient that most closely matches yours. When the background turns green, move on to the “Next Conflict” and repeat until they’re all resolved and then click on “Apply Changes” (bottom left of the window).

Add the protein layer by dragging the protein from the sidebar into the A1 well of your new plate. Save the experiment, print a label, and you’re done!

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