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FlyChip protocol for adding spotting buffer to 384-well printing plates

Overview

The oligonucleotide libraries were dispatched to us in 384-well microtitre plates. Spotting buffer was added to the plates using an MWG-Biotech Roboseq 2500 Liquid Handling Robot (LHR).

Equipment and reagents

• MWG-Biotech Roboseq 2500 Liquid Handling Robot
• 384-well microtitre well printing plates with the long oligonucleotides
• 70% Ethanol
• Spotting buffer made using distilled water
• Hettich Rotina 35 microtitre plate centrifuge
• Adhesive PCR Film (Abgene; Cat. No. AB-0558)
• Horizontal laminar flow work station (Jencons; Cat. No. 566-031)

Procedure

Before using the LHR

1. Store the spotting buffer 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 clone-set 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 clone-set.

Whilst using the LHR

6. Clean tips using at least 3 cycles of the "Flush.rss" wash program. Once completed, open the LHR script you created during step 5.
7. 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.
8. Load spotting buffer 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.
9. Check if position 6 needs more spotting buffer, if so re-fill and then restart the program and follow the on-screen intructions.
10. 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

11. Centrifuge all plates at 2000 rpm for 5 minutes to ensure the spotting buffer 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.
12. Centrifuge all plates at 2000 rpm for 5 minutes to ensure the spotting solution is at the base of each well and then store all of the plates at -80 °C until they are next required.

R. Auburn (17-02-2006).