Firma:              Idaho

Modell:            2380

Kommentar:    Dokumente engl.

The Rapidcyclerm is a rapid tempeeture cycling system based an heat transfer by hot air to samples contained in thin capillary tubes. For most reactions, products can easily be visualized with ethidium bromide an agarose gels after a total reaction time of 15-30 minutes. The rapid temperature response of this instru­ment can improve product specificity significantly while decreasing the required reaction time by up to an order of magnitude.

Average temperature transition rotes in most instruments are commonly about 1° C/sec when metal blocks or water are used for thermal equilibration and samples are contained in plastic microfuge tubes. A significantfraction of the cycle time isspent heating and cooling the sample, as opposed to being spent at optimal temperatures. Long reaction times of 2-6 hours are com­mon, and slow transition rotes make it difficult to deter­mine optimal temperatures and times for each stage of the cyclic reactions. Instantaneoustemperature changes are notpossible because of sample, container and cycler heat capacities, "Second generation" instru m entation can complete 30-cycles in about one hour. lt is unlikely any instrument based an samples contained in conical tubes with heating and cooling through a metal block can achieve faster cycles at any price. However, with capillary tubes and air heating, transition Tates of 5-10°C/ sec are easily obtained. Complete 30-cycle reactions can be finished in os little as 10 min. Biochemical reactions are fast. The Rapidcycler is the first instrument engineered to match this speed.

The advantages of an air-cycling system include simplicity, low cost, and rapid temperature cycling. Air is an ideal heat transfer medium which can change tem­perature quickly because of its low density. Temperature homogeneity problems are solved by rapidly mixing air with a fan to provide homogeneous temperature expo­sure over the sample containers, The sample container is just os important os the heat transfer medium. An optimal sample container should be water-vaportight and have:

(i) low thermal mass,

(ii) good thermal conductivity,

(iii) minimal internal condensation,

(iv) easy sample recovery without cross contamination,

(v) No adverse effects an the reaction.

Whatever the containermaterial, temperatureequilibra­tion will always be achieved faster if the sample volume is small, if the container wall is thin, and if the surface-to­volume ratio of the sample exposed to the container wall is high. Problems with condensation can be reduced by minimizing the free air space surrounding the sample.

vicrofuge tubes are kept water-vapor tight by mechanical closure and, if necessary, overlaid mineral oil. Thermal conductivity is poor because of the material and its thickness (about 1 mm). Internal condensation can occur if mineral oil is not used and particularly if different parts of the tube are at different temperatures.

Sample mixing by convection has been used in conical tube Instruments, The temperature gradients that cause convection are not a good Idea for a temperature­dependent reactions

Capillary tubes are kept vaportight by flame clo­sure of the ends, They conduct heat to the sample better than microfuge tubes because of decreased wall thickness (ca. 0.2 mm) and a better surface-to-volume ratio. Dead air space is minimized to prevent significant condensation, Different volumes of a sample can be placed in the same diameter capillary tube so that rapid heattransfer is maintained,

After amplification, the ends of the glass capillaries can be quickly scored with a fite and snapped off with less risk of aerosolization and contamination than microcentrifuge tubes. The capillary tubes nerve both os a transfer pipette and a container for temperature cy­cling, The reaction product, already containing Ficoll and an electrophoresis indicator dye, can be directly emptied into a gel well without exposureto an intermedi­ate pipette tip or to extraction procedures.

Decreasing the hegt capacity of the cycling sys­tem can markedly decrease the total time required for reactions that require temperature cycling, In addition, air cycling and miniaturization can significantly decrease the costs of reagents and the personnel time required to optimize reactions,