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Effects of Thermal Cycler Well-To-Well Temperature Uniformity on PCR Results

the RoboCycler system. These fluid temperature results show that PCR cycling conditions using the RoboCycler 96 cycler are virtually identical from well to well.

PCR Results: RoboCycler vs. MJ Research PTC-100

Experiments were performed to determine if, and to what extent, the temperature variability observed across the block of the MJ Research PTC-100 during fluid temperature testing affected PCR product formation. A fragment of the trkA gene was PCR-amplified from a cDNA library made from human brain frontal cortex. A single PCR master mix was prepared large enough to accommodate six reactions for each of the two thermal cyclers tested. The 50-l aliquots from this master mix were placed into 12, 200-l thin-walled tubes. Six of these tubes were positioned in a RoboCycler 96 temperature cycler fitted with Stratagenes Hot Top Assembly, and six were positioned in an MJ Research PTC-100 with hot top enabled. As described for the fluid temperature measurements, all four corners and two center wells were included. The annealing temperature chosen was known to produce several PCR products that could be used for comparison. Following thermal cycling, aliquots from each reaction were run on a vertical 6% polyacrylamide/TBE minigel, stained with ethidium bromide, and visualized using the Eagle Eye II Still Video System (Figure 3).

Figure 3

The yield and specificity of PCR products obtained using the RoboCycler 96 were similar for all wells tested. However, PCR results obtained using the MJ Research PTC-100 showed a significant degree of well-to-well variability. Similar results were observed when using the RoboCycler standard paddle and the MJ Research PTC-100 with the
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