Fig 1.
Photograph of the thermocycler device.
The device is 40 cm long x 25 cm wide x 23.5 cm tall from the bottom of the enclosure (feet excluded) to the top of the camera system.
Fig 2.
Two views of the hot blocks, cassette, and cassette positioning system.
Four hot blocks and a cooling block (on the right) are shown. A stepper motor, lead screw and cassette carrier move the cassette from block to block.
Fig 3.
Single hot block with temperature sensor.
Fig 4.
Schematic representation of the thermocycler system.
Fig 5.
It is machined from a single piece of aluminum and has 10 trenches (grooves) for holding sample containing capillaries.
Fig 6.
Three views of a PCR cassette.
(a) an empty cassette, (b) a cassette loaded with 50 capillaries, and (c) a cassette packaged for storage.
Fig 7.
Cross-sectional view of the PCR cassette with capillaries and wax.
(a) capillaries where the gel is desiccated before the sample is added. (b) capillaries after the sample has been added and the gel has hydrated, but before the wax is melted. (c) capillaries after the wax has melted after the cassette was heated during the PCR.
Fig 8.
Schematic representation of the epifluorescence system.
Fig 9.
Photographs of the cassette during melt curve data collection.
These images were taken during the MCA after 35 cycle PCR with stx2 primers. The corresponding melt peaks are shown in Fig 12.
Fig 10.
Plot of hot block and cassette temperatures during one cycle of PCR.
(a) Pre-denature, denature, anneal and extend block control temperatures and block temperatures, and cassette temperature. (b) Pre-denature and denature block control temperatures and block temperatures. (c) Extend block control temperature and block temperature. (d) Anneal block control temperature and temperature. i) Cassette temperature rise when moved to pre-denature block. ii) Cassette temperature fall when moved to anneal block. iii) Cassette temperature rise when moved to extend block. iv) Pre-denature block control temperature change to pre-bias temperature. v) Slow rise of pre-denature block temperature when the cassette is away from the pre-denature block. vi) Constant denature block control temperature and block temperature. vii) Pre-denature block temperature fall when cassette arrives at the pre-denature block. viii) Pre-denature control temperature change to working temperature when the cassette stops on the pre-denature block. ix) Steady pre-denature block temperature while the cassette rests on the pre-denature block. x) Extend block control temperature change to pre-bias temperature. xi) Slow rise of the extend block temperature when the cassette is away from the extend block. xii) Quick rise in the extend block temperature when the cassette passes over on the way from the denature block to the anneal block. xiii) Extend block temperature fall when the cassette stops on the extend block. xiv) Extend control temperature change to working temperature when the cassette stops on the extend block. xv) Stable extend block temperature while the cassette rests on the extend block. xvi) Anneal block control temperature change to pre-bias temperature. xvii) Slow anneal block temperature fall while the cassette is away from the anneal block. xviii) Anneal block temperature rise when the cassette stops on the anneal block. xix) Anneal block control temperature change to working temperature when the cassette stops on the anneal block xx) Stable anneal block temperature while the cassette rests on the anneal block.
Fig 11.
Real-time PCR for the amplification of stx2 gene in diluted overnight culture of E. coli.
PCR was performed for (a) 35, (b) 30, and (c) 25 cycles. The curves show fluorescence amplitude versus number of PCR cycles. The second row shows the CCD images at the 35th, 30th, and 25th cycle for each PCR, respectively. Capillaries in trenches 1–8 were hydrated with the diluted culture of E. coli with dilution factors of 101, 102, 103, 2x103, 104, 2x104, 105, 2x105, and 106 respectively. Trench 10 was hydrated with water.
Fig 12.
MCA curves for the PCRs performed with (a) 35, (b) 30, and (c) 25 cycles (shown in Fig 11).
Fig 13.
MCA curves of the five capillaries in the entire trench for the three highest dilution factors.
(a) 105, (b) 2x105, and (c) 106 for the PCRs performed with 35 cycles (from Fig 11). MCA curve of one of the negative capillaries is plotted to show the baseline.
Table 1.
Detection of 1–3 bacteria using the GelCycler Mark II as determined by a Poisson distribution/fractional analysis of positive capillary reaction units.