A1: PCR on the Spartan DX^TM can be performed as either Real-Time analysis or Endpoint analysis. In Endpoint analysis, the fluorescence of the last cycle is compared with the fluorescence of the first cycle. In Real-Time analysis, the fluorescence is measured after each cycle of PCR. The threshold cycle (Ct) is the cycle when the fluorescent signal exponentially increases above the baseline fluorescence. A lower Ct value indicates a higher concentration of starting DNA template compared to a higher Ct value. E.g. a Ct of 5 indicates a higher starting concentration of DNA than a Ct of 15. Therefore, Endpoint analysis is ideal for qualitative DNA detection experiments (yes/no), whereas Real-Time analysis is ideal for quantitative or semi-quantitative experiments where you are interested in the relative quantity of the starting DNA material.

A2: We recommend dyes, probes, and fluorophores that have excitation peaks below 520 nm and emission peaks between 500-580 nm. Examples include SYBR® Green I dye and fluorophores such as FAM, TET, and Cal Fluor Gold® 540. These fluorophores should be coupled with quenchers that do not fluoresce, such as Black Hole Quenchers® (BHQ). FRET probes cannot be used on the Spartan DX^TM because the donor dye (usually fluorescein) has a very high baseline fluorescence and masks the emission signal of the fluorophore.

A3: A kit with an incorporated reference dye cannot be used. Many kits contain a passive reference dye (usually ROX) that is required to adjust the fluorescent signal correctly. The Spartan DX^TM has a single detection range that works well with most fluorophores and does not need to be corrected for signal intensity. The use of a reference dye will interfere with the proper detection of your fluorophore of interest.

A4: 20 µl is the ideal reaction volume, although volumes from 10-50 µl may be used. Larger volumes decrease the efficiency of the heating and cooling kinetics and longer temperature dwell times may be required (see Application Note 008).

A5: We recommend thin-wall tubes from Fisher Scientific (Cat. No. 08-408-214) and DiaMed (Cat. No. AD0210-FCN) (see Application Note 017).

A6: For real-time PCR, SYBR® Green final concentrations typically range between 0.1 to 1.0X. We have found that concentrations of 0.2-0.7X work well on the Spartan DX^TM, and 0.4X works the best (see Application Note 009). Avoid using concentrations of 0.8X or higher because this may decrease PCR efficiency.

A7: SYBR® Green I is a light-sensitive dye. It is also sensitive to freeze/thaw cycles and is less stable when diluted in water. To minimize light degradation, store the dye in a dark place at 4-8°C. To minimize freeze-thaw degradation, split the stock dye into multiple aliquots and store in a dark box at -20°C. Once thawed, a 10 000X aliquot may be stored in a dark box at 4-8°C for up to 3 months. At 4-8°C, dilute stocks of 100X are stable in water for a few days at and 10X stocks are stable for 1 day. For commercial kits that have been freeze-thawed and have decreased SYBR Green fluorescence, consider adding some extra SYBR Green dye to the mix (See FAQ #6, and see Application Note 009).

A8: Mixing and spinning the tubes may give more precise results, but the reagents are usually mixed well by thermal convection during the denaturation step.

A9: The solution may be to pick a shorter probe system (such as MGB probes) rather than a longer system (such as TaqMan® probes). The reason is because shorter probe systems hold the fluorophore and quencher closer together, and thus enables the quencher to quench the baseline fluorescence more effectively. Reducing the background fluorescence improves the curves generated by the Spartan DX^TM.

A10: An isolated spike in fluorescence may be caused if the PCR tube is not properly seated or if there is debris in the well. The reason is because these conditions cause the tube to shift slightly when it is being imaged by the optical module. This shift in position causes a light reflection which then results in a spike in fluorescence. To prevent this problem, make sure that the tubes are always properly seated in the machine. Also, consider using thin-wall flat cap tubes from Fisher Scientific (Cat. No. 08-408-214) and DiaMed (Cat. No. AD0210-FCN) that we have validated for use on the Spartan DX^TM. If there is debris lodged in a well, try cleaning it out (see FAQ #13).

A11: The Spartan DX^TM requires a 10 minute warm up period to allow the internal cavity of the machine to reach a steady temperature. If the machine is not allowed to reach a steady temperature prior to use, you may observe variations in threshold cycle (Ct) values or even failed reactions.

A12: The Spartan DX^TM has been designed for operating temperatures ranging from 19°C to 30°C. Operating the machine at higher temperatures may prevent the low-temperature heat block from being maintained at a sufficiently cool temperature.

A13: We recommend cleaning the machine’s temperature blocks once every 6 months. To do this, set both block temperatures at 40ºC and then pause the machine on the hot block. Then, use cotton swabs soaked with 70% ethanol to individually clean out each of the four wells. Once cleaned, then dry the wells with a cotton swab. To prevent dust and debris from falling into the machine, we recommend keeping the lid closed at all times.

A14: If you have any problems, please contact us any time by email at support@spartanbio.com or by phone from 9am-5pm EST at +1 [877] 228-7756.

A15: A conventional three-temperature PCR protocol has three steps: denaturation, annealing, and extension. A two-temperature protocol combines the annealing and extension steps into a single step.

Step (1): Check that the melting temperatures of your primers are greater than 58°C (for the Spartan DX^TM, optimum melting temperatures are between 62-68°C). Since most DNA polymerases have optimal enzymatic activity around 72-76°C, the combined annealing/extension step should be set around 55°C or higher. An annealing temperature of 55°C or higher should work if your primers have melting temperatures of 58°C or higher. If your primers have melting temperatures lower than 58°C, the reaction may still work. If it does not, then you may need to redesign them so that they have higher melting temperatures. Primers with melting temperatures of 65°C and higher are particularly well suited for faster programs.

Step (2): If you know the melting curve profile for your primers, pick a denaturation temperature that is to the right side of the curve. If you do not know the profile, then start with a denaturation temperature of 93°C.

Step (3): Now that you have picked annealing and denaturation temperatures that are appropriate for your primers, use the Spartan DX^TM “Temperature Selection Chart” to determine the temperature settings to program into the Spartan DX^TM.

Step (4): Set up your PCR reaction mixture and run on the Spartan DX^TM, using the two-temperature program determined in Step (3).

A16: The Spartan DX^TM does not have a hot lid. Therefore, we recommend adding 15 µl of mineral oil (e.g. Sigma-Aldrich, Cat. No. M8662; Biotools, Cat. No. 20.032) to the top of your reaction mixtures to prevent evaporation. After adding the oil, you may wish to spin down the reaction tubes in order to get even distribution of the oil over top of the reaction liquid.

A17: The primers should be designed so that their melting temperatures fall within the range of 62-68°C.

A18: The limitation is that primer melting temperatures may need to be higher for two-temperature programs than for three-temperature programs. The advantage is that much faster run times may be achieved with two-temperature programs because the annealing and extension steps are combined into one step.

A19: We recommend the Primer3 program (http://frodo.wi.mit.edu/) because it enables you to set variables such as melting temperature, minimum and maximum primer lengths, % GC content and salt concentrations.