One temperature cycler can meet the needs of an entire laboratory
Michelle Cayouette Jane Moores Jason Capodanno
Michael Steege Michael Rahilly
Stratagene is introducing the RoboCycler Infinity temperature cycler,# a new temperature cycler with interchangeable blocks for PCR. Block sets can be readily interchanged without the use of tools and are currently available in a 40-well, 600-l tube format and a 96-well, 200-l tube format. A 384-well block set will be available in 1998. Each block set is available with an optional Hot Top Assembly, which reduces condensation and allows oil-free PCR amplifications. PCR experiments showed similar yield and specificity of amplified products regardless of the block format or paddle type used. As with all members of Stratagenes line of RoboCycler temperature cyclers, the blocks of the RoboCycler Infinity model showed a high degree of well-to-well uniformity.
In 1992, Stratagene introduced the RoboCycler temperature cycler, a unique four-block instrument that achieves unparalleled temperature uniformity.1 Using a robotic arm, the RoboCycler unit moves tubes from one preset hot block to another, avoiding the longer reaction times associated with ramping the block temperature of single-block thermal cyclers. Another feature of the RoboCycler temperature cyclers is the built-in cold storage block that allows experiments to be run conveniently overnight.
With the introduction of the Hot Top Assembly in 1996, the RoboCycler temperature cycler became even more versatile. The Hot Top Assembly heats the top and sides of reaction tubes, significantly reducing condensation and eliminating the need for oil overlays on samples.2,3
The new RoboCycler Infinity temperature cycler incorporates all of the features of a standard RoboCycler unit with the flexibility of interchangeable block formats. The RoboCycler Infinity cycler consists of the base unit, removable blocks and removable paddle assembly (figure 1). Without using tools, researchers can change block formats by removing the self-contained paddle assembly from the main unit of the RoboCycler Infinity cycler, unplugging the block modules and installing a different block and paddle set. Currently, the block sets are available in 40-well, 600-l tube and 96-well, 200-l tube formats. A Hot Top Assembly is available for both of these block formats so that the RoboCycler Infinity cycler can provide oil-free PCR amplifications. As a safety feature, the RoboCycler Infinity temperature cycler contains a power cut-off switch, which turns off power to the block module connectors when a block is removed. All other aspects of the RoboCycler Infinity cycler, such as well-to-well uniformity, user interface and programming, robotic operation and physical dimensions are identical to other RoboCycler models.
Since the RoboCycler Infinity temperature cycler features interchangeable blocks, the specificity and yield of a PCR product should be equivalent regardless of which block format is used during an experiment. PCR amplifications were performed to compare the pattern and yield of amplified products obtained using either the 40-well or 96-well block formats.
In these experiments, primers specific for the human glucocerebrosidase gene were used to amplify human genomic DNA. Reaction components and PCR parameters were as previously described.3 A large master mix was prepared, and 50-l aliquots were added to six thin-walled tubes. The tubes were positioned in the four corner wells and two center wells of the 40-well paddle of the Hot Top Assembly. A second set of reactions was prepared and cycled using identical PCR parameters and similar well positions using the 96-well block format and hot top assembly. Although not optimal for this primer-template system, an annealing temperature of 60C was used to allow both specific and extraneous PCR product bands to be compared. In addition, small deviations from 60C have been shown to produce discernible differences in banding patterns.3 After cycling with both block formats, 15-l aliquots of each reaction were electrophoresed through VisiGel separation matrix, stained with ethidium bromide and visualized using the Eagle Eye II still video system. Comparable product specificity and yield were observed when using either the 40- or 96-well block format (figure 2). Well-to-well variability was also low for both block formats, indicating that uniform temperature was maintained across the blocks during the amplifications. Similar results were observed with a different primer-template system (data not shown), indicating that consistent PCR results are obtained regardless of the block format used during experiments.
PCR amplifications were performed to compare the product yield and banding pattern generated using either the Hot Top Assembly or standard paddle. The glucocerebrosidase primer-template system was again used for these PCR amplifications. Reactions were amplified using the 40-well block format and Hot Top Assembly. The Hot Top Assembly was then changed to the standard paddle and amplifications were performed as with the Hot Top Assembly, except that an oil overlay was added to each reaction. As in the first experiment, multiple reactions were aliquoted from the same master mix and positioned throughout the blocks during each run to test the well-to-well variability. After cycling was complete, 15-l aliquots from each reaction tube were electrophoresed, stained and visualized as before. In this experiment, comparable yield and specificity of products were observed using either the Hot Top Assembly or the standard paddle and an oil overlay on reactions (figure 3). Similar results were obtained using the 96-well block format (data not shown). In all cases, well-to-well variability was low. These results demonstrate that the addition of the Hot Top Assembly to a RoboCycler Infinity temperature cycler fitted with either the 40- or 96-well block format did not adversely affect the formation of PCR products or well-to-well temperature uniformity.
The interchangeable block RoboCycler Infinity temperature cycler allows 200-l or 600-l tubes to be run on a single machine. Block sets are currently available in a 40-well and a 96-well format. In addition, a 384-well block set is in development and slated for a 1998 release. The blocks and paddle assemblies are easily and quickly exchanged without the use of tools. PCR results obtained using the RoboCycler Infinity temperature cycler showed similar product yield and specificity regard less of the block format or paddle type used. The new RoboCycler Infinity cycler preserves the unique four-block design, robotic operation, run speed and well-to-well uniformity that are common to all RoboCycler units.
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