Hydrodynamic DNA Shearer
High Capacity Shaker
Template Preparation Machine
Flow Through Micro-Centrifuge
Plasmid Preparation Machine
|Template Preparation Machine
We have developed an instrument for automated purification of ssDNA from phage
supernatant based on a glass fiber purification protocol. The instrument is served by two server
arms. The input arm serves microtiter plates containing M13 and e-coli pellets to the instrument by
retrieving them from our standard 14-plate cassettes. The instrument
collects 300 ul of phage supernatant from the input plate,
mixes it with precipitation buffer and transfers the mix
into one of six Polyfiltronics GF/B filter plates which are
mounted on the instrument. After all six plates have been loaded, the instrument
simultaneously performs the wash protocol outlined below on all six plates. During these washes,
the phage are trapped in the filter membrane and lysed, and the DNA is released, bound to the
glass, and washed with ethanol. Once the washes are complete, the pipettor arm of the instrument
resuspends the clean DNA in 1xTE and elutes it into a collection plate served by the second server arm.
The instrument produces approximately 50 ul of ssDNA per well at approximately 80 ng/ul.
At this time, it can process six microtiter plates in 1 hr 20 min, though improvements are planned
to increase throughput to six plates per hour and to perform e-coli cell separation in the
instrument itself. The cost of the prep is currently $0.12/well and is dominated by the cost of the
filter plate. Re-use of the filter plates is possible, lowering the cost of the prep if desired. This
instrument has been in production use since 1/96 and is now producing an average of 25,000
templates per month for our sequencing projects. All ssDNA templates made at our center since
early 1996 have been made with this instrument. PCR product purification is also occasionally
performed with this machine operating with a slightly different protocol.
ssDNA purification protocol performed by the instrument:
- Mix 300ul of M13 supernatant with 80uL of 20% PEG 8000 + 2.5M NaCl. Transfer to
glass fiber filter.
- Push liquid through filter under pressure to trap phage in filter.
- Wash filter twice with 3M NaClO4 in 70% EtOH to lyse phage and bind DNA to
- Wash filter six times with 70% EtOH to remove salts.
- Dry filter with compressed air.
- Add 60 ul of 1xTE to each well. Incubate for two minutes.
- Elute into microtiter plate.
This instrument is now available through Genemachines.
For information call them at 650-508-1634
or e-mail them.