Genome Technology Center

Template 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:

  1. 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 glass.

    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.

  2. Elute into microtiter plate.

This instrument is now available through Genemachines. For information call them at 650-508-1634 or e-mail ( ) them.

Template Preparation Machine
Template Preparation
Sample Loading Movie
Input Server
Input Server Image
Input Server Movie
Supernatant Collection
Supernatant Collection
Aspiration Movie
More Sample Loading
More Sample Loading
Sample Transfer
Sample Transfer Image
Sample Transfer Movie
Wash Cycle
Underside of Filter Disc & Waste Tray
Pressure Filtration Movie
Filter Disc Rotating
Elution Buffer Transfer
Drip Director Drying
Filter Disc
Filter Disc
Disc Rotating Movie
Template Collection
Template Collection Movie
Output Server Arm
Output Server Arm
Another Image
Server Arm Movie
Pressure Station
Pressure Station Image
Pressure Station Movie

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