Workshop
Protocols

Soybean
EST Project

1. Soybean cDNAs from the EST project have been cloned in either pSPORT 1 (Gibco/BRL) or pBluescipt®II SK (+) (Strategene) plasmid vectors in DH10B host cells.
2. Each 384-well plate of a bacterial library is split into four 96-well plates to grow the bacteria and isolate plasmids (done by Keck Center using 96 well transfer devices).
3. Plasmids are purified by Keck Center using a Qiagen robot to obtain high quality DNA templates. These templates are used for sequencing of the 3' end. The concentration of plasmid DNA is typically about 100+ ng/ul. The same template DNAs are also used for PCR reactions to obtain the PCR for microarrays after dilution as described below.
4. Dilute plasmid DNA to a concentration of 0.5-2 ng/ul using 1 ul of template per 100 ul of sterile nanopure water.
5. This is done either with a robotic pipettor or in the following manner.

   - Pour sterile nanopure water into a sterile trough. Use 10 mls per 96 well plate.
   - Use a Matrix electronic 8-channel pipettor to add 100 ul to all wells of four empty 96 well storage plates (Evergreen from Phenix: catalog #LMP-8001).
   - Use a Finnpette manual 8-channel 0.5-10 ul pipettor to transfer 1ul of plasmid prep (from the Keck Center's 96-well plates containing the high concentration Qiagen DNA plasmids) to the 96-well plates just filled with water.

6. Mark plates as Diluted DNAs for PCR and approximately 1 ng/ul concentration.
7. Cover platles securely with 4-inch wide aluminium foil tape. Spin at 2000 rpm for 1 minute and store at -20°C.