Solid-phase reversible immobilization (SPRI) for the purification of PCR products (96-well format).

This protocol came from the Whitehead Institute's web site and is based on the paper by Hawkins

Run 50 ul PCR reactions using standard methods in 96 well plates. For plasmids, we use 2-3 ul of 2-3 hour culture in a PCR reaction.

Prepare beads: Wash 10 mg/ml carboxyl coated magnetic beads three times with WASH BUFFER (0.5M EDTA (pH 8.0)). The beads come 10% solids which is 100 mg / ml. So dilute 1/10 in 0.5 M EDTA. Apply magnet and remove buffer. Repeat three times. Leave washed beads in 0.5 M EDTA.

For each PCR reaction (50 ul), add 10 ul of washed beads and 50 ul of HYB BUFFER (2.5M NaCl/20% PEG 8000). Mix well and incubate at RT for 10 min.

Place the microtitre plate on a magnet for 2 min and wash the beads twice with 150 ul of 70% EtOH. Air dry for 2 min, and resuspend the beads in 20 ul of ELUTION BUFFER (10mM Tris-Acetate (pH 7.8)) and incubate at RT for 5 min.

Magnetically separate the beads and remove the supernatant for testing and sequencing.

The SPRI PCR method binds DNA based upon size. We have shown previously that the lower limit at which yields in excess of 80% are achieved is 200bp, the maximum limit is in excess of 200Kb (BAC DNA isolation).

Overall this solid-phase procedure is fast, simple and highly automatable.
Need information on what (if anything) differs for plasmid DNA

KC 3/02