High Throughput Robotics Facility

Opened in 2002 by the vice chancellor Professor Robert F. Boucher CBE, FREng, the automated equipment was originally used in a research programme to identify novel regulatory proteins involved in inflammatory signal transduction.

For further information contact:
Karen Holland/Luke Marsden
Telephone: 0114 226 1484
Email: k.holland@sheffield.ac.uk or l.marsden@sheffield.ac.uk

Liquid Handling on Q-bot

Q-bot is a highly customised pneumatics operated robot that is able to run a variety of liquid handling routines on 96- and 384- well plates and incorporates a number of specialised features:

• Two 96 tip liquid handling heads to enable volumes of1-50ul and 5-250ul to be accurately aspirated/ dispensed
• Rows and columns option to allow head to use only 8 or 12 tips respectively (option only available to accurately aspirate/ dispense volumes of 5ul and over)
• Two vacuum systems that allow for a vacuum of 8"Hg to be pulled from the top section of the manifold or 24"Hg to be pulled from the bottom
• Two Camlab Teleshakers that allow for liquid shaking in 96- or 384-well plates. Speeds between 100 and 2,000rpm can be utilised with a gradual start of the shaking process to eliminate sample splashing. Shaking movements that can be selected are circular, linear or diagonal
• UV light source for sterilization of the robot deck
• Robotic deck that can hold 24 plates (not including those on teleshakers, vacuum systems and lid removal station). A maximum of 4 different plate types can be used in 4 sets of 6
• CO2 incubator that can contain up to 40 plates
• Plate hotel that can contain up to 20 plates
• Automated plate handling and lid removal/replacement to allow for sample sterility (only available for plates removed from the hotel or incubator)
• Specialist data tracking system, with barcodes read using a CCD camera and relayed to LIMS
• Specialist computer software allowing for any type of 96-/384-well plate to be used on the robot, including deep well blocks

Liquid Handling Routines

Q-bot has a high throughput capacity and can be used for a number of procedures including the following:

• Transfections
• Minipreps
• PCR set up
• DNA extraction
• Aliquoting into 96-/ 384-well plates
• Expansion of libraries stored in 384 well plates to 96 well format
• Compression of libraries in 96 well plates to 384 well format to save storage space
• Replication to deep well blocks

If there are any liquid handling routines not mentioned above that you would like to run on Q-bot, please contact Karen Holland or Luke Marsden

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MegaPix

MegaPix is a high speed automated picking system designed around a highly accurate linear system (x- and y- axis) and ball screw system (z- axis) to combine speed precision and flexibility of picking selection criteria. It can be used for both colony picking and re-arraying, being both precise and cost effective when compared to manual picking.

MegaPix comprises of an enclosed working area into which plates and Qtrays are automatically delivered. Colonies or clones are picked by a 96 pin picking head that is thoroughly sterilised after each inoculation.

The room in which MegaPix is situated has the option of being cooled to 10oC to minimise colony and clone growth.

Features

• Unique barcode reading system enabling plates to be validated and logged
• Rigorous pin sterilization procedure incorporating an ethanol wash bath and a hot air pin dryer to prevent sample carry over. The number of bath cycles, drying times and wait times (to allow pins to cool) can be altered to suit individual requirements
• UV light for enclosure sterilization
• Choice of pin fire order
• Plate stackers can hold up to 70 plates
• Qtray stackers can hold up to 50 Qtrays
• 96- / 384- well plate option, with choice of plate type. If a particular plate type is not already in the system, it can be added by the operator
• When using 384- well plates, the wells of each quadrant to be inoculated can be specified
• Choice of the number of dips to inoculate the destination plate
• Choice of the number of pins to be used before each inoculation
• Choice of inoculation time

Colony Picking on MegaPix

Colony picking using MegaPix is an automated process involving the transfer of colonies from bioassay trays into pre-filled well plates containing broth media.

MegaPix is capable of picking in excess of 4,000 colonies per hour and eliminates errors associated with manual picking. This is made possible by utilizing an integrated vision, detection and analysis system, integrated with novel software and a 96 pin picking head.

Colonies or plaques are grown in Qtrays (22.2 x 22.2cm2 trays) ideally to a size above 0.5mm for efficient picking. Once placed into a stacker system, these are delivered to the robot bed and the colonies or plaques are imaged using a CCD camera. Qtrays have been specifically designed for this type of application as they offer high clarity for enhanced resolution and discrimination of colonies.

The unique picking software automatically selects the colonies that fall within the limits set by the user-defined criteria and shows the summary. For each colony, a pin fires from the picking head, touches the surface and retracts. When all (or a pre-selected number) of the pins have been fired, the head inoculates sterile medium in 96- or 384- well plates. This continues until all selected colonies have been picked.

Run Options

• A duplicate set of plates can be produced
• Manual selection / de-selection of colonies
• Ability to define total number of Qtrays to be processed per run
• Ability to define first picking pin
• Ability to reserve certain pins from firing so wells can be used for control data
• Choice of minimum/maximum colonies to be picked per region of the Qtray. If the number of colonies is unavailable in a region, the shortage can be made up from another region or the plate can be padded with blanks
• After each region has been picked, option of inoculating in next well offset or subsequent plates

Colony Selection Preferences

• Diameter – maximum / minimum area of colony in pixels
• Roundness – ratio of perfect circle taken form measured radius of colony divided by actual circumference of colony
• Colour – colour or grey value of colonies to be picked. This is white for normal colonies, blue/white if white colonies picked on blue/white plates or phage
• Proximity – distance to neighbouring colonies
• Axis ratio – shortest axis divided by longest axis
• Halo colonies – nucleus ratio may be used to set the percentage ratio between colony and halo area
• Exclusion zone – distance from edge of Qtray that is excluded from colony selection

Re-arraying on MegaPix

Re-arraying or cherry picking is the re-distribution of previously collected samples into new 96- or 384- well plates. This is easily achieved when using a robotic system such as MegaPix and is quicker and less error-prone than re-arraying manually.

After screening a clone library, it is likely that it contains several putative positive clones that are needed for a secondary screen or to be prepared for DNA sequencing. Rather than working with several plates from the original library, each one containing only one or a few clones of interest, it is much more practical to re-array these in to as few plates as possible.

The plate and well co-ordinates for each positive clone can either be inputted into the specialist software or recorded in a user-defined database. The first picking pin is selected and a choice of whether to stir the source well is given. During the process, barcodes can be validated using the on-board CCD camera, eliminating plate stacking errors. Each time a re-arraying sequence is run, an activity log file is generated recording details such as run time, source wells picked and their respective destination positions.