APPENDIX E

Picking clones using the Genetix QBot

(a) Setting up the QBot for picking: Before picking is initiated, the UV lamps should be used to sterilize the working area in the QBot. After UV-sterilization, the wash bath should be filled with 70-100% ethanol, and one or two X/I/C trays (without lids) placed in appropriate holders on the light table. 384-well microtiter plates containing sterile freezing medium should be placed in the hotel(s), and the hotel(s) should be properly mounted in the QBot. The "Picking" software application should be opened, and the camera calibrated. The instrument is now ready to begin its automated picking tasks.

(b) Clicking on the "scan" icon begins the automated phase of the picking cycle. The camera on the robotic arm is positioned over an X/I/C plate. Starting in a specific corner of the tray, the arm makes a series of discrete movements so that all sectors of the X/I/C tray are eventually scanned by the camera. The image of each tray sector is captured by the QBot.

(c) After an image of a particular sector of the tray has been captured, the QBot's picking software analyzes the image and selects objects that meet the characteristics of single, spatially-isolated, white colonies. On the monitor, objects that meet the necessary criteria automatically are circled in green. Objects that meet some, but not all of the criteria are circled in red. Only colonies circled in green will be picked. The user can "fine tune" the selection process for a particular tray by changing the thresholds of various parameters including colony roundness, relative color, and size (this is usually done before automated scanning is actually initiated). After the X/I/C tray(s) has been completely scanned and evaluated by the picking software, the QBot is ready to begin picking.

(d) Once the picking cycle is initiated, the QBot's first order of business is to sterilize the picking pins. In brief, the robotic arm places the bottom of the picking head (i.e., the pins) into the ethanol wash bath. The arm makes a series of short, jerky movements effectively moving the pins back and forth through the bath. Groups of clustered bristles lining the bottom of the bath enhance the cleaning process.

 

(e) Upon completing the wash/sterilization cycle, the arm places the picking head into the blow drier. The warm air released by the blow drier causes evaporation of residual ethanol left on the pins.

 

(f) The QBot moves to the front of the hotel. The first 384-well plate (plate 1) is gently grasped by the plate gripper, and removed from the hotel.

 

(g) The robotic arm places the plate on the "loading dock" of the plate holder. The gripper releases the plate, and the arm moves away from the plate holder.

 

(h) The plate holder moves the plate forward so that it is in "working position". The lid manipulator rotates 180° so that it is directly over the plate. The manipulator drops onto the plate lid. Using four vacuum-controlled suckers, the lid manipulator attaches itself to the lid. The lid is lifted off the plate, and the manipulator rotates back to its original position taking the lid along with it.

 

(i) The lid manipulator places the lid on a "low profile" resting pad adjacent to the plate holder. The manipulator remains attached to the lid.

 

(j) The picking head is moved into position over the X/I/C tray. Because the plate has already been scanned, and the exact locations of suitable clones determined, the picking head immediately begins picking clones from the tray. One by one, each of the 96 pins is fired into a different colony. After penetrating a colony, the fired pin is quickly retracted. If the instrument is properly calibrated, most if not all of the pins will hit the proper target colonies.

 

(k) Once all the pins on the picking head have been fired, the picking head is moved into position over the 384-well plate. The head is lowered so that each of the 96 pins is inserted into one (and only one) well of the plate. Presumably, some of the bacteria adhering to each pin are dislodged from that pin when it enters the freezing medium. Consequently, the pins "inoculate" the wells. Inoculation only takes a couple of seconds. The robotic arm then moves the picking head up and away from the microtiter plate. Because there are 384 wells in a microtiter plate and only 96 picking pins, (384 ÷ 96 =) 4 rounds of picking and inoculation are required to completely inoculate a single 384-well microtiter plate.

 

(l) Once the first plate has been completely inoculated, the lid manipulator puts the lid back on the plate. The plate holder shifts the plate back into "transport position" and the plate is picked up by the plate gripper on the robotic arm. The inoculated plate is placed back into the same slot of the hotel from which it was originally removed.

 

(m) The robotic arm and plate gripper remove the second microtiter plate from the hotel, and the process of picking and inoculating plates continues.

 

 Return to CONTENTS

Go on to APPENDIX F