CHAPTER 16

Once the library (or a portion of the library) has been plated, clones are picked and placed into freezing media in microtiter plates to create an ordered BAC library (see ordered libraries). Each suitable white colony is placed into a single well of a microtiter plate either by hand (using sterile toothpicks) or with an automated picking system.

After clones have been plated and allowed to form colonies, it is important to pick and store clones within the next 14 days. To produce an ordered library, white colonies are transferred from an X/I/C plate into freezing medium in the wells of a microtiter plate. The inoculated microtiter plates are incubated overnight. The "master copy" of the library is then used as a template for producing replicate copies of the library (see CHAPTER 17).

Clones can be picked by hand using sterile toothpicks or by an automated colony picker. As discussed in CHAPTER 2, there are several companies that currently make colony-picking robots. We use the Genetix QBot for picking as well as other library-related tasks. However, less expensive picking instruments are available, and there are several companies and non-profit organizations that offer picking/replicating/gridding services (see CHAPTER 2). While an in-depth discussion of robotic colony pickers is beyond the scope of this paper, an overview of the principles behind automated picking is presented in the section below.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): Freezing medium; sterile 384-well or 96-well microtiter plates; a multi-channel pipettor, a repeat pipettor, or an automated plate filling system (e.g., the Genetix QFill2)

METHODS:

1. Fill sterile 96-well or 384-well plates with freezing medium. If 96-well plates are used, place 150 µl in each well. If 384-well plates are used, place 60 µl of freezing medium in each well. Plates can be filled with hand-held pipettors (preferably multi-channel pipettors or repeat pipettors). However, the easiest and fastest way to fill plates is using an automated plate-filling device such as the Genetix QFill2 (use of the QFill2 is illustrated in APPENDIX C). The number of plates that should be filled will depend upon the desired genome coverage and the number of clones available.
2. Label each plate with regard to organism/genotype, date of library construction, quality designation, and plate number. Plate labels should be placed on the edge of the plate at the "frosted end". An example of a plate-labeling scheme is shown in FIGURE 16.1.
3. Once microtiter plates containing sterile freezing medium have been prepared, picking can begin. Two methods of picking clones are discussed in section II below.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): X/I/C trays with clones (see CHAPTER 15); sterile (autoclaved toothpicks) or a hand-held colony picker; 384-well or 96-well plates filled with freezing medium (see I above)

METHODS:

1. Place 10-20 filled and labeled microtiter plates, a box of sterile toothpicks (or a hand-held colony picker; see CHAPTER 2 for details), and one of the X/I/C trays in a sterile laminar flow hood.
2. Wash hands with soap and water. Spray hands with 70% ethanol and wipe dry using a Kimwipe.
3. Open the box of sterile toothpicks, and take the lids off the first microtiter plate and the X/I/C tray.
4. Pick up a sterile toothpick by one of its ends. Carefully stab one of the white colonies on the X/I/C tray with the other end of the toothpick. Make sure that the toothpick only touches one clone.
5. Place the toothpick in one of the wells of the microtiter plate. Leave the toothpick in this well so that there is no doubt that this well has been inoculated (FIGURE 16.2).
6. Repeat steps 4 and 5 until all wells in one row have been inoculated (i.e., all wells contain a toothpick). Carefully remove the toothpicks one at a time from the plate (removal of several toothpicks at once increases the likelihood of cross-contamination between wells). Place the "used" toothpicks in a biohazard bag or autoclave them for re-use.
7. Continue until all of the wells of the plate have been inoculated. Place the lid back on the microtiter plate and set it in the corner of the hood away from the other microtiter plates.
8. Continue the process until you have filled all of the microtiter plates necessary to meet your desired genome coverage and/or you have picked all the useable clones off of your X/I/C plates.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): X/I/C trays with clones (see CHAPTER 15); sterile (autoclaved toothpicks); 384-well or 96-well plates filled with freezing medium (see I above); a QBot (Genetix) or a similar high-throughput genomics robot

METHODS: Most automated colony picking robots have a similar mode of operation. In general, one or more X/I/C trays are placed in the machine. Lids are removed from the trays. A camera system scans the trays, and the robot’s internal computer system uses various algorithms to determine what objects scanned by the camera meet the characteristics of a white colony. The computer’s decisions are based upon the relative roundness of an object, its proximity to other objects, and its color relative to the background. The parameters governing the robot’s decisions can be adjusted by the user. Once the tray(s) has been scanned and clones have been selected for picking by the computer, a picking head (composed of a series of pins/needles) is positioned by a robotic arm over the surface of the plate. Each pin is fired into one of the "pre-selected" colonies. After all the pins in the head have been fired, the picking head is moved into a position over an open microtiter plate containing freezing medium. The pins are used to inoculate wells on the plate. The pins are then sterilized and the picking cycle is repeated until the microtiter plate has been completely inoculated. A diagram of the Genetix QBot is shown in APPENDIX D. An overview of how the QBot picks clones is given in APPENDIX E.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): sterile (autoclaved toothpicks); inoculated microtiter plates (see II above)

METHODS:

1. Carefully wrap stacks of the inoculated microtiter plates with plastic wrap. These plates constitute the master copy of the library. Place the plates in a 37ºC incubator for 14-16 hours.

­ Note 16.1: Use of a water-jacketed incubator prevents evaporation from the plates and thus eliminates the need for plastic wrap.

2. Remove the master plates from the incubator. For a given plate, the media in each well should appear turbid due to bacterial growth. If the plates were prepared using an automated picker, it is probable that some of the plates will contain a well or two in which no bacteria have grown (possibly due to a bent or broken pin). If desired, these empty wells can be inoculated by hand and the plates can be placed back into the incubator for an additional 12 hours. Plates without any empty wells should be re-wrapped in plastic and left at room temperature.

   ­ Note 16.2: Do not leave plates at room temperature for more than two days. Proceed with replication (CHAPTER 17) as soon as possible.

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