Stanford
Genome Technology Center
Technology
Development Group
| Instruments
Thermocycler |
Thermocycler
Commonly available thermocyclers implement a 'hot-plate' design. That is a metal plate which is designed to accept a 96-well micro-strip. This plate is actively heated and cooled to cycle through the appropriate temperatures (MJ Research) While such designs have proven reliable they do suffer from a few fundamental limitations. First, sample volume must be above some minimum (usually approx 10µl) for the reactions to proceed reliably. Second, in the case of the MJ, thermal cycling is achieved by heating and cooling a massive metal block. The heat capacity of the block slows the temperature ramping time. The design which we have chosen to implement requires few moving parts and avoids the time lags associated with changing the temperature of large masses. It is based on a device built by Scott Hunicke-Smith in which thermal cycling is achieved, not by changing the temperature of a block, but by moving the sample through regions of the appropriate temperature. In this conceptually simple design we assemble three copper blocks, each with 1/16" diameter holes drilled through in a standard 96-well format, in a stack such that the arrays of holes co-align. Each of the blocks is maintained at extension, annealling, or denaturing temperature, and the blocks are well insulated from each other. Each hole is threaded through with a teflon tube through which the samples will move. One end of each tube is extended above the block assembly to connect to the syringe needles of a Robbins Scientific Hydra-96 pipettor. The other end extends below the assembly for loading the samples from a standard 96-well plate. By activating the Robbins pipettor we can easily load samples, and move them through all three temperature stages. The thermocycler is designed to interface easily into our automated system; the plates are stored in a cassette and carousel facility, and may be loaded to the thermocycler using our standard server-arm mechanism. TechnicalThermal stability of the copper blocks is maintained by using commercially available temperature controllers (Newport Electronics). Each block has heating elements (Ni-chrome wire) on the top face, bottom face, and around the perimeter, and each heater has a dedicated controller. This use of three temperature controllers to regulate the temperature at each face ensures maximum homogeneity of the thermal profile.The Teflon tubes have several desirable properties: smooth surfaces allow smooth sample movement, hydrophobic surfaces ensure that the sample slug stays intact, and the inherent chemical stability of Teflon virtually eliminates the possiblity of contamination. The Robbins Scientific pipettor allows precise sample movement and its 96-channel format is ideal for our application. We modified our instrument to allow computer control through a serial port and we have written routines in LabView to control the movement and timing for processing sequencing reactions. Future PlansWe have modified the current implementation to include the use of another servo motor which will move the entire thermal block assembly. In this upgraded design (shown on the figure) we load the sample into the tubes and then move the temperature stages past the samples, while holding the samples in place in the Teflon tube. This modification allows highly precise positioning of the samples relative to the thermal blocks, and faster cycling times because the speed of movement for the blocks is not limited in the same way as is movement of the fluid slugs. |
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