CHAPTER 17

Once the master copy of the library has been made, it is important to make one or two replicates of the master as quickly as possible. Replication can be performed using a hand-held replicator or an automated plate replicator (e.g., a QBot). Once replicates have been made, the master copy can be stored at –80ºC indefinitely. The Quality I (QI) replicates can then be used to make Quality II (QII) copies of the library, and QII copies can be used to make QIII working copies of the library (see quality designations for a discussion of the meaning of QI, QII, QIII, etc.). Once QIII copies have been made, the QI and QII copies are stored at –80ºC as well. QIII plates are used to make BAC grids to which DNA probes of interest can be hybridized.

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: For every microtiter plate in the master copy of the library, fill one or two microtiter plates with sterile freezing medium (see CHAPTER 16). Whether you prepare one or two plates depends on whether you want to make one or two QI copies of the library. Label the plates appropriately (see FIGURE 16.1)

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): 384-well or 96-well microtiter plates filled with sterile freezing medium; a hand-held, reuseable plate replicator; the master copy of the library (CHAPTER 16)

METHODS:

­ Note 17.1: Perform replication in a sterile laminar-flow hood.

1. Sterilize the replicator.

1. If a stainless-steel replicator is used, dip the pins of the replicator into a 70% ethanol bath. Remove the replicator from the alcohol bath. Ignite the residual alcohol on the pins by passing the replicator through the flame of a Bunsen burner. Allow the pins to cool for two minutes.
2. If a plastic (polypropylene) replicator is used, wash the pins thoroughly in 12% v/v bleach. Rinse the pins thoroughly with MBG water followed by a quick rinse in 95% ethanol. Allow the pins to air dry.

2. Perform replication as shown in FIGURE 17.1.
3. Repeat steps 1-2 until all of the plates in the master copy have been replicated either once or twice.
4. Proceed to III.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): 384-well or 96-well microtiter plates filled with sterile freezing medium; a QBot (Genetix) or a similar high-throughput genomics robot; the master copy of the library (CHAPTER 16)

METHODS: Plate replication using a high-throughput genomics robot is based on the same principles as manual plate replication (see FIGURE 17.1). However, a robot rather than a human(s) performs plate manipulation and inoculation tasks. An overview of how the Genetix QBot replicates BAC libraries is given in APPENDIX F. Once replication is complete, proceed to III below.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): the master copy of the library; any other copies of the library that are ready for long-term storage; library storage boxes

METHODS:

1. Arrange the microtiter plates containing the master copy of the library in stacks of nine plates. For simplicity, place plates 1-9 (from top to bottom) in the first stack, plates 10-18 in the second stack, etc. (FIGURE 17.2a).
2. Placing plates in storage boxes:

1. If cardboard storage boxes are being used, carefully bind each stack of plates with two rubberbands as shown in FIGURE 17.2a. Be careful not to sharply jar or tilt the plates as this will result in cross-well contamination. Place the first four stacks of plates (i.e., plates 1-36) in a library storage box as shown in FIGURE 17.2b. Close and label the box as shown (FIGURE 17.2c). Place the next four stacks of plates in a second box and label accordingly, etc.
2. If metal storage boxes are being used, load plates into the metal boxes as shown in FIGURE 17.3.

3. Once the master copy of the library has been placed into storage boxes, place the boxes containing the master copy into a –80°C freezer.
4. Repeat steps 1-3 for all library copies that you want to place in long-term storage.

Go back to CONTENTS

Go on to APPENDIX A