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FlyChip
Functional Genomics for Drosophila
Cambridge Systems Biology Centre, Tennis Court Road, Cambridge, CB2 1QR, UK  [map]
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Library desiccation and rehydration

Overview

Spotting solution within 384-well microtitre plates evaporates during printing. This evaporation is uneven, wells at the edge and especially in the corners evaporate faster than those in the centre. This is best overcome by desiccating and rehydrating the entire library. Water is added to the 384-well microtitre plates using an MWG-Biotech Roboseq 2500 Liquid Handling Robot (LHR).

Equipment and reagents

Procedure

Desiccation

  1. Remove all of the printing plates (384-well format) from the -80 °C freezer and leave to thaw on a desk.
  2. Take note of which 384-well microtitre plates the DNA is stored in (this information is required later).
  3. Switch the laminar flow work station on and leave for 30 minutes before use, i.e., as per the manufacturers instructions.
  4. Once defrosted, centrifuge all plates at 2000 rpm for 5 minutes to remove excess moisture and to ensure that the probe DNA is in the base of each well.
  5. Remove the seals and leave the plates in the laminar flow work station until all wells in all plates have been desiccated (e.g. two to three days).
  6. Once desiccated, the plates should then be resealed and can be stored at -80 °C before continuing with the rest of this protocol.

Before using the LHR

  1. Store the water that will be used to rehydrate the plates at 4 °C to ensure that it is chilled prior to use.
  2. Clean the exterior and interior of the LHR using the Dyson vacuum cleaner and then wipe with 70% Ethanol.
  3. Replace the LHR flush water with a fresh batch. Clean tips using at least 3 cycles of the "Flush_Long.rss" wash script. This will perform 400 tip flushes per script run and will remove any contaminants.
  4. Test the LHR to ensure that it is performing correctly by transferring water to 384-well microtitre plates of the type the library is in. The test script to use is called "384_disri.rss".
  5. Evaluate and then save the LHR script under a different name. This is the script that will then be used to add water to the library.

Whilst using the LHR

  1. Clean tips using at least 3 cycles of the "Flush.rss" wash program. Once completed, open the LHR script you created during step 11.
  2. Remove all the microtitre plates from the -80 °C freezer and then leave to thaw. Once thawed, centrifuge all plates at 2000 rpm for 5 minutes to remove all surface moisture.
  3. Load water in the cooled rack (position 6). Then start the script and follow the on-screen instructions:
    • Plate seals should be removed as the plate is being loaded into the LHR
    • All plates should be loaded in the LHR such that well A1 is in the top-left corner of each plate position
    • An experienced person should be present and observing the LHR at all times to ensure correct functioning of the instrument.
    • As each plate is finished with, remove from LHR, seal with adhesive PCR film, then store at 4 °C.
  4. Check if position 6 needs more water, if so re-fill and then restart the program and follow the on-screen intructions.
  5. Once the run is completed the microtitre plates can be stored at -80 °C until they are next required. Re-clean the LHR to ensure the LHR is left in a reasonable state for others to use.

Rehydration

  1. Centrifuge all plates at 2000 rpm for 5 minutes to ensure the water is at the base of each well. Then place all of the plates at 37 °C for 2 hours to ensure the probe DNA is redissolved and the spotting buffer reformed.
  2. Centrifuge all plates at 2000 rpm for 5 minutes to ensure the water is at the base of each well and then store all of the plates at -80 °C until they are next required. Ensure the FlyChip internal data tracking system is updated to record that the library has been subjected to this process.

R. Auburn (04-04-2005).