Workshop
Protocols

Soybean
EST Project

1. Place the sephadex measuring plate (MultiScreen â Column Loader) on a clean piece of saran wrap. Pour some sephadex onto the plate (takes about 3.3 g). Scrape the surface with the Multiscreen metal plate with the plastic scraper so that the sephadex will fill all 96 wells. Place Polyfiltronics 96-well filter plate on top of filled measuring place. Hold together very tightly and flip such that the sephadex falls out of the wells of the mesuring plate into the Polyfiltronics wells. Keeping plates lined up, tap the measuring plate with a marker pen so that all the sephadex comes out. Remove the measuring plate and brush off the bottom of the Polyfiltronics plate with a clean brush and place it on top of a clean piece of aluminum foil.
   
   
2. Add 300 µl of sterile water to each well of Polyfiltronics plates with the Matrix electronic 8-channel pipettor. Cover with a piece of parafilm. Incubate for 4 hours at room temperature. After 4 hours, place it on top of a 96-well plate 500ul microtiter plate (Evergreen from Phenix, catalog #LMP-8001. Secure with rubber bands and spin them down at 750x g for 90 sec.
   
   
3. Remove the microtiter plate (these can be re-used until they break) and replace it with a new, labeled MJ Research polypropylene 96-well (200ul) PCR plate (catalog #MSP-9621) underneath. Secure them with rubber bands at both ends.
   
   
4. Remove the plates containing the PCR products from the refrigerator. Spin them at 4000 rpm for 1 min. Add samples (should be ~50 µl in each well) very carefully (try not to touch the sephadex) to the center of the sephadex unifilter using a Finpette manual 12-channel pipettor.
   
   
5. Spin at 750x g for 90 sec. Remove the sephadex unifilter plates and dry down the sample plates either overnight in a tissue culture hood or for one hour in a speed vac.
   
   
6. Add spotting solution which contains10 µl of 3X SSC with or without 0.05% Sarkosyl (we are still indecisive on whether to use sarkosyl) using the Finnpette manual 0.5-10µl 8-channel pipettor. Seal the plates with a 4-inch wide aluminium foil tape and store them in the refrigerator.
   
   
7. Prior to use, vortex plate and spin at 4000 rpm for 1 minute.

Parts to be supplied:

MultiScreen ® Column Loader: Millipore, Catalog no: MACL 09645

Sephadex from Amersham Pharmacia; G-50 DNA grade F: Catalog no: 17-0573-02

Polyfiltronics Unifilters (from Fisher): Whatman ® Polyfiltronics, Unifilters ® 96 wells, Cat no: 7700 - 3304