COLONY ARRAYING WITH THE VIRTEK CAPS SYSTEM

version: 1.1

You will need

Set-Up

Arraying Yeast The CAPS system will transfer yeast between liquid culture in microtiter plates and agar plates, or between plates of the same type. Currently, we only possess 384-pin replicators for the CAPS. Individuals using the system must initially create an agar plate of yeast arrayed in a 384-patch format using a hand-pinning tool or a 384-well microtiter plate of yeast cultures using the MultiMek? or similar pipeting tool.

Machine Start-up

Compressed Air Supply The compressed air supplies the pneumatic controls of the plate gripper arm, attached to the left of the Z-axis. Use the regulator on the tank (at the back left of the machine, yellow tank, thin blue plastic outlet line) to bring the air pressure to 100 psi. Once the system is pressurized, the gripper arm should rise to its maximum height, away from the surface of the table. Always perform this step first to ensure that the gripper arm does not collide with anything once the motors begin moving.

Vacuum Valve This is the small tan box to the left of the cabinet, which controls the vacuum used for aspirinating wash solutions. It should always be switched on, as indicated by the red light on the front of the box.

Check Vacuum Trap The flask on the table surface serves as the vacuum trap for aspirated liquids. Empty the trap if there is any liquid in it.

Check Humidifier Water Level At the back right of the machine, on the floor, is a lab ice bucket containing the pump for the humidifier. This should be completely submerged in water (tap water is OK). Additionally, the outflow line should be return to the same reservoir, to capture the water that comes out of the humidifier.

Air Filter The power switch for the air filter is found on the left side of the cabinet, high up, towards the back. The air flow into the filter is controlled by the intake port on the left side towards the back. To increase the air flow, slide the metal sheet to expose more of the port. Air will not begin flowing until the humidifer is switched on also.

Humidifier Turn on the humidifier using the switch on the right side of the cabinet, high up, towards the back. Once the air filter and humidifier are switched on, you should be able to feel a gentle flow of air coming out of the cabinet at the gaps in the door. This does not need to be a strong flow of air, as just a little positive pressure will prevent microorganisms from entering the cabinet, but should be detectable. The humidity level in the chamber should be around 55% -- too high and the pins will stick in the head and puncture your agar, too low and the plates will dry out and your yeast will not be as happy.

Embedded Controller The motors of the robot are powered by the embedded controller, housed in a large cubic box directly underneath the table. The front of the box has a small door that flips open to reveal a round power button. Press this button to turn on the embedded controller. You should hear a beep, then a click from inside the cabinet.

Note: There is also a power switch and a circuit breaker on the back of the embedded controller -- please use the switch on the front to turn the power on and off.

Computer Boot up the computer that is under the table towards the right side.

un: Administrator pw: yeast

Software Launch the CPCA.exe application from the shortcut in the center of the Desktop. Select your user name from the list and click OK.

Running the Software

Console You can launch the Console by clicking its button on the toolbar: it looks like a small computer with a big “C” on its screen. This is the window you use to run scripts on the instrument and to interact with the command line. The window should open automatically when the program in launched.

Homing It is necessary to home the motors before any other commands are issued to ensure that the motion does not take the machine beyond its limits. Click the Homing button in the top center of the Console window. You will only be able to home the system once each time it is turned on.

Joystick Controls The simplest way to move the motors around, either to get the head away from the working surface or to recover from a bug, is to use the Joystick window. Launch this window with the button on the toolbar that looks like a plane with an arrow rising vertically from it. Clicking a button once will move the selected axis a specified distance -- clicking and holding a button will cause the selected axis to jog at a defined speed. Three levels of distance/speed are available. Be careful not to move the motors beyond their physical limits. Also be certain that the replicator head is clear of obstructions on the deck when executing an X-Y move.

Control Panel Just as the Joystick window can be used to control the motor axes, the control panel can be used to control all the non-motor axes. Launch this window using the button with the two button icons on it. The gripper arm can be opened and closed, and the vacuum line can be activated. Press C5 empty to turn on the vacuum line for this system.

Writing a Script

The methods that control the CAPS are called Scripts and are composed of two parts, a Configuration and a set of Tasks.

Configuration

This tells the CAPS what is where on the deck of the instrument. The window will display a view of the deck, including the Elevator, the Wash Positions, and the Frames (?). To change what is in each section, you must first click on it -- the bottom half of the window will change to reflect what labware, rinse basins, etc., can be placed in those positions.

Elevator We do not have the elevator option installed on our CAPS.

Wash Positions There are five wash positions on the left side of the deck. Two are specially modified spots on the deck and three are generic. The special ones are a sonicator and heated fan for drying -- their positions on the deck cannot be modified.

The other wash positions simply consist of basins that need to be manually filled and emptied, using the aspirator. A basin of this type corresponds to a “Wash 3” designation in the pulldown menu. Place one of these in each of the 3 available wash positons.

Platforms Platforms hold the labware in specific locations so that the head can be used accurately. Several kinds of platforms are available -- we only have Type 1 platforms, which have space for 10 plates. These two platforms should be flush against one another and pushed completely to the left of the deck.

Plates The CAPS can handle rectangular Nunc OmniTrays? and other standard microtiter plates. Add these plates to the layout by selecting a plate type from the pulldown menu at the bottom of the window. You will need to select the radio button for Platform/Stacker to get access to the plates. Clicking the Gel Plate radio button will place the standard formats for agar plates into the pulldown menu. Once you have selected the right labware, click on a platform location to place the labware there.

Wash settings Set these by clicking on “Wash Settings” and selecting appropriate order from the pulldown menus. The standard washing procedure should go:

Use sterile water for the washes. The wash solutions should be changed every cycle. If cell debris is a problem, it can be dramatically reduced by placing a sponge in the first wash basin, to help shear away the cells from the ends of the pins. The sponge should be placed in the first wash basin and then submerged in sterile water (fill the chamber until the sponge is only just fully submerged, otherwise you may overflow the chamber when the pins push down on the sponge). The sponges used are from VWR (catalog number 58542-246, 5-1/2” x 3-5/8” x 1-1/8”). They need to be trimmed to fit the basin: 7.5 cm x 12.5 cm x 1.5 cm thick (just cut each to half its usual thickness with a scalpel).

Tools Click on this button to launch the tool selection window. Most choices are not selectable, as we do not have those options installed on our instrument. The one tool that we currently have is a Replicator 384. Check that box and close the window. This is an easy step to forget!

Lids Be very careful when selecting labware to enter into the configuration: make certain you indicate a lid if there is one and do not indicate a lid if there isn’t one. Both situations will cause you problems.

Saving

When you are finished laying out the Configuration, click the Save As button and give the file a name. You will use the Tasks interface to tell the software what to do with the described configuration. The file will be saved with a .script extension.

Tasks

This is a list of what the robotic arm is actually going to do with the plates you have layed out on the platform. Lauch the task editor window from the toolbar window.

Load a Configuration The software is designed to greet you with an error when this window is launched. Do not be alarmed -- click OK. Two windows should be opened, a configuration map (which will be empty) and a task editor. The first step is to load a configuration by clicking on the Load Configuration button in the Task Editor window. Select the .script file that you saved when you configured the platform above.

Add Steps Steps must be added to the method in order. You cannot insert or delete steps from the middle of the method list. Be careful to enter the correct settings for each step -- if you make a mistake early, you will have to go back and delete all the steps after that.

Transfer Clicking this button changes your cursor into a white syringe-ish icon. First, click on the Configuration window on the plate to transfer from. Second, click on the plate to transfer to. Third, adjust the settings in the window that pops up.

Dip Number Where in the colony the robot will pick from. Positions 5/0 correspond to the center of the colony, 1 is the back-left, and 9 is the front-right, and so on counting left-to-right, then top-to-bottom.

Dip Level How far down the replicator will go down into the plate, in mm.

Replicates How many times to touch the agar, i.e. 0 will do it just once, 1 will do it twice, and so on.

Wash Step? Check this box to perform the washing protocol that you entered into the configuration when the transfer step is finished.

Dips A transfer step is really just two dip steps with a wash step afterwards. If you want to do something more complicated, e.g. dip onto more than one destination plate, you can use the individual dip steps instead of a transfer.

Washes If you do use some Dip steps, don’t forget to follow them with a Wash step. All washes will use the standard wash configuration that you enter in the Configuration window unless you change it.

Saving Scripts

The tasks are automatically saved when you click the OK button in the task editor window. They are saved to the same .script file that houses the configurations. Any changes you make to the configuration will obliterate your tasks.

Running a Script

After you have created a list of tasks for your platform configuration, return to the Console window to run them.

Open Tasks Use this button to select your .script file. Once the file is open, the list of tasks should be visible in the window in the upper left-hand corner of the window.

Place Labware on Platform Place all of your plates on the deck in the locations given in the configuration. Be very careful to place the plates pushed into the appropriate corner -- i.e. away from the springs in the position. The springs are not strong enough to force the plate into the corner, so you need to give each a nudge.

Fill the Wash Reservoirs Make sure there is clean liquid in each of the wash reservoirs, in accordance with your wash protocol. To remove dirty liquid, use the house vacuum line attached to the aspiration flask. Yeast will settle out of the solutions and collect on the bottom of the reservoir -- be sure to remove these as well. The reservoirs lift off the deck easily.

Command Line

There are a number of commands that are very important for the operation of the CAPS, but have no buttons to send that command to the robot. Therefore, you will need to learn these fun commands.

The standard wash protocol takes about 2.5 minutes to run, so doing a whole 10 plate to 10 plate mating should take less than an hour.

Shutting Down

Simply go through the start up process above in the reverse order.

Ordering Information

Hand Pinning Tool V&P Scientific, Inc. (800) 455-0644 VP 408FH MULTI-BLOT E-CLIP FLOATING PIN REPLICATOR - 96 PINS

$990

Guide for Hand Pinning Tool V&P Scientific, Inc. (800) 455-0644 VP 381 LIBRARY COPIER - REGISTERS REPLICATOR TO MICROPLATES $75

Nunc Omni-Tray, with lid Nalge Nunc International 242811

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