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Oil-Free PCR Using the Hot Top Assembly

Hot Top Assembly now available for Robocycler 96 temperature cyclers

Michelle Cayouette Jane Moores Michael Steege Jason Capodanno Michael Rahilly
Stratagene Cloning Systems, Inc.

With the introduction of the Hot Top Assembly for RoboCycler 96 temperature cyclers, researchers can begin running oil-free PCR amplifications. Each Hot Top Assembly for RoboCycler 96 cyclers is supplied with a software upgrade card, which provides more accurate temperature control. Hot Top Assemblies are available as options on newly purchased RoboCycler temperature cyclers.# Individual units that can be added to existing RoboCycler 40 or 96 units are also available. Using both gradient and single-temperature RoboCycler 96 models, we show the yield and specificity of PCR products to be equivalent before and after the addition of the Hot Top Assembly and software upgrade. Furthermore, PCR results at a given annealing temperature were reproducible whether the annealing block was run in gradient or single-temperature mode, an important feature for optimizing annealing temperatures. When used for small-volume cycle sequencing experiments, the Hot Top Assembly did not affect results adversely, and gel loading was quicker and easier without oil or wax overlays on reactions.

Stratagenes line of RoboCycler temperature cycler currently includes four models. The RoboCycler 40 temperature cyclers accommodate up to forty 600-l individual PCR tubes; the RoboCycler 96 units are high-throughput models that accommodate either a 96-well plate or ninety-six 200-l individual or strip tubes. Both the RoboCycler 40 and 96 temperature cyclers are available in either gradient or nongradient models. Gradient models facilitate PCR optimization by allowing researchers to simultaneously test multiple annealing temperatures. All members of the Rob oCycler family feature a unique four-block design and a robotic arm that moves a paddle containing PCR tubes from block to block at user-specified times. The standard paddle of the main unit of the RoboCycler temperature cycler can be replaced by the Hot Top Assembly, which is an independently powered unit.

RoboCycler units fitted with Hot Top Assemblies heat reaction tubes from the top and sides as well as the bottom. This uniform heating dramatically reduces the amount of condensation that forms during cycling and eliminates the need for oil or wax overlays on PCR reactions. Because the Hot Top Assembly features an automatic shut-off function and over-temperature sensor, PCR amplifications can be run safely overnight. Individual at-temperature and power-on indicator lights provide a quick visual appraisal of the instruments status. The Hot Top Assembly is now available either as an individual unit that can be added to any existing RoboCycler units or as an option on newly purchased RoboCycler 40 or 96 temperature cyclers.1

Each purchase of a Hot Top Assembly for the RoboCycler 96 temperature cycler includes an upgraded version of the software that controls the main unit. The software PC card containing the updated software is simply inserted into the PC card slot located on the right side of the RoboCycler main unit, adapting any RoboCycler 96 temperature cycler for use with the Hot Top Assembly. The updated version of the software comes installed on new RoboCycler 96 temperature cyclers purchased with the Hot Top Assembly option. The upgraded version of the software provides more precise temperature control when using the Hot Top Assembly and, in the gradient model, maintains a linear, 11C temperature gradient across the annealing block, from a minimum temperature of 45C up to a maximum temperature of 99C. In single-temperature mode, the annealing block maintains a stable temperature bet ween 37 and 99C. RoboCycler 40 temperature cyclers that are equipped with Hot Top Assemblies do not need an upgraded version of software to maintain precise temperature control.1

Comparison of PCR Results: Before and After Addition of a Hot Top Assembly

figure 1

Experiments were performed using a RoboCycler Gradient 96 temperature cycler* to determine if replacing the standard paddle with the Hot Top Assembly and installing the software update PC card would alter the yield and/or specificity of PCR products. Human genomic DNA was used as the template in a PCR amplification with primers specific for glucocerebrosidase, the gene associated with human Gauchers disease. An 11oC temperature range was generated across the gradient block, providing annealing temperatures from 49 to 60C. Each well in a row differed from the preceding well by 1C. A PCR master mix was made, and tubes containing 50-l aliquots of the mix were placed into wells that corresponded to each of the 12 different annealing temperatures in the gradient range. Reactions were cycled under the appropriate conditions on a RoboCycler Gradient 96 temperature cycler fitted with the standard paddle and the original software. The standard paddle was then replaced with a Hot Top Assembly, and the software was upgraded using the PC card. A new PCR master mix was made, aliquoted into tubes and cycled as before. After both experiments were complete, equal volumes of the PCR products were electrophoresed through the same gel, stained with ethidium bromide and visualized using the Eagle Eye II still video system (figure 1). Equivalent banding patterns were observed when using either the original configuration of the RoboCycler unit or the retrofit configuration. Therefore, the addition of the Hot Top Assembly did not interfere with the formation of an 11C gradient across the annealing block. Experiments performed on a nongradient RoboCycler 96 as well as nongradient and gradient RoboCycler 40 temperature cyclers also showed similar, consistent results when either the standard paddle or the Hot Top Assembly was used (data not shown).

Using the Gradient Mode to Optimize Annealing Temperatures

Figure 2A

figure 2B

RoboCycler Gradient 40 and 96 temperature cyclers aid in experimentally determining optimal annealing temperatures by providing a linear temperature range across the gradient, which allows simultaneous testing of up to 8 or 12 different annealing temperatures.2-5 To optimize an annealing temperature, researchers must be able to choose a temperature from a gradient experiment and consistently reproduce results during subsequent experiments with the annealing block in single-temperature mode. PCR experiments were performed to determine if addition of the Hot Top Assembly to a RoboCycler unit alters the capacity for optimizing annealing temperatures. Reactions were set up and run sequentially, in either gradient or single-temperature mode, on a RoboCycler 96 temperature cycler fitted with a Hot Top Assembly and updated software. As before, human genomic DNA served as the template with primers specific for the glucocerebrosidase gene. The gradient was set to maintain a temperature range from 49 to 60C, and the experiment in single-temperature mode was performed with an annealing temperature of 54C. For the single-temperature portion of the experiment, 10 aliquots from a large master mix were positioned throughout the block (figure 2B) in order to test well-to-well consistency. Following the gradient and single-temperature runs, aliquots of PCR products were electrophoresed through the same gel, stained with ethidium bromide and visualized using the Eagle Eye II still video system (figure 2A). Banding patterns from the 54C single-temperature annealing experiment closely matched those observed for the corresponding temperature tested within the gradient range, indicating that the Hot Top Assembly did not diminish the ability to optimize annealing temperatures using the gradient block. In addition, the Hot Top Assembly did not alter the well-to-well consistency when using the gradient block in uniform mode.

Cycle Sequencing Using the Hot Top Assembly

Unlike conventional PCR experiments where reaction volumes of 25 to 100 l are common, cycle sequencing experiments are performed using very small reaction volumes. Consequently, cycle sequencing is likely to be sensitive to small amounts of liquid lost from reactions during cycling because this amount makes up a larger percentage of the original reaction volume than in a standard PCR experiment. Cycle sequencing also requires small volumes of radioactive liquid under oil overlays to be removed from tubes for loading onto a sequencing gel. For these reasons, cycle sequencing is a good experimental test of the ability of the Hot Top Assembly to decrease condensation formed during cycling and to preserve the quality of results obtained using the standard paddle.

figure 3

Duplicate cycle sequencing reactions were set up and performed under identical cycling parameters on a RoboCycler 96 temperature cycler. The first set of reactions was performed with the standard paddle and an oil overlay. The standard paddle was then removed and replaced with the Hot Top Assembly. The second set of reactions was prepared with the oil overlay omitted and cycled using the Hot Top Assembly. After cycling, equal volumes of the sequencing products were electrophoresed on a sequencing gel using the CastAway system.6 The gel was fixed, rinsed with water, dried and exposed to film. When either the standard paddle or the Hot Top Assembly was used, equivalent cycle sequencing results were obtained (figure 3). Similar results were observed when the experiment was performed using a RoboCycler 40 temperature cycler (data not shown).

Conclusions

By heating PCR tubes from both the top and sides, the Hot Top Assembly for RoboCycler temperature cyclers dramatically reduces the amount of condensation formed during cycling and eliminates the need to add oil or wax overlays to reaction tubes. The Hot Top Assembly makes postcycling manipulations, such as gel loading, significantly faster and easier, especially when many samples are analyzed. Our results demonstrate that the addition of the Hot Top Assembly to RoboCycler units did not compromise the quality of PCR or cycle sequencing results when compared to those obtained using a standard paddle and an oil overlay. By installing the new software upgrade PC card, which comes with the Hot Top Assembly for the RoboCycler 96 unit, any RoboCycler 96 temperature cycler can be retrofitted to become an oil-free PCR machine. Hot Top Assemblies are now available for any RoboCycler 96 or RoboCycler 40 temperature cycler, either as separate units that can be added to existing machines or as an option on newly purchased cyclers.

REFERENCES
  1. Cayouette, M., Moores, J., and Rahilly, M. (1996) Strategies 9: 75-76.

  2. Danssaert, J., et al. (1994) Strategies 7: 2.

  3. Danssaert, J., et al. (1994) Strategies 7: 66-67.

  4. Cayouette, M., et al. (1995) Strategies 8: 58-60.

  5. Cayouette, M., Gackstetter, T., and Moores, J. (1996) Strategies 9: 33-35.

  6. Giambalvo, J., and Hedden, V. (1996) Strategies 9: 39-40.


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