Real-time Thermal Cycler FAQs

Q. What is the excitation light source used in PrimeQ?

A. It is a white LED with an excitation range of 470-650 nm.

Q. What is the detector used in PrimeQ?

A. A single photomultiplier tube with a detection range of 500-710 nm.

Q. What is the dynamic range of detection of PrimeQ?

A. At least 9 orders of magnitude depending on the assay.

Q. How long does PrimeQ take to scan a plate?

A. The scan time required for a 96-well plate depends on the integration or dwell time. The integration time is the length of time the fibre optic stops over each well to collect fluorescence data. The default integration time is set to 150ms which means that it will take approximately 20s to read all 96 wells. For chemistry methods with a strong signal to noise ratio and a brighter fluorophore, the integration time can be reduced to as little as 50ms per well, reducing the scan time to approximately 10s. Conversely, integration time can also be increased to a maximum of 250ms per well; in this case the background or noise will also increase proportionally. In addition, when programming, there is also an option to read only filled wells which reduces the scan time even further if not all 96 wells are being utilised. In the latter case, a plate layout needs to be defined before the run to tell the instrument which wells to read.

Q. What filter sets are currently available for PrimeQ?

A. There are four filters supplied with PrimeQ (FC02-FC05) which cover the range of most available fluorescent dyes. The optional filter, FC01 is also available for multiplexing with certain dyes. The excitation and emission wavelengths are as follows:
FC01: ex 460nm, em 500nm
FC02: ex 485nm, em 520nm
FC03: ex 530nm, em 560nm
FC04: ex 580nm, em 615nm
FC05: ex 640nm, em 685nm

Q. When would I use filter FC01 instead of FC02?

A. FC01 can be used for multiplexing FAM™ with dyes such as Yakima Yellow®, TET™ and JOE™ (when read with FC03) with no cross-talk. We generally recommend using HEX™ for multiplexing with FAM; this is because HEX has excitation/emission wavelengths of 535/556nm. These wavelengths are longer than those for YY, TET and JOE (531/549, 521/536 and 529/555 respectively) which means there is greater spectral separation from FAM. By reducing the wavelength we use to measure FAM with the FC01 filter, we can increase the separation from the excitation/emission wavelengths of the other dyes and so avoid any cross-talk. See Application Note A08 002A: PrimeQ filters for further information.

Q. What sample volume can I use in PrimeQ?

A. Techne recommends using 20µl reactions in PrimeQ as this works well and is economical; however volumes as low as 10µl have been tested and give satisfactory results (see Application Note A08 001A: Minimising Sample Volumes for further details). At the higher end, 50µl reactions can be used where greater sensitivity is required.

Q. Is PrimeQ real-time PCR machine 21 CFR part 11 compliant?
A. No, because like most qPCR machines, Techne PrimeQ is primarily intended for research. Research and in-vitro diagnostics are not normally involved with submissions to the FDA. qPCR machines complaint to 21 CFR 11 are quite niche and expensive.

However, although not compliant, PrimeQ and Quansoft software answer the most important concern of 21 CFR 11 because all results files are encrypted, and can not be altered by the user. Furthermore, no data created outside of Quansoft (e.g. MS Excel) can be imported back into Quansoft. In this way, Quansoft data analysis and presentation functions are totally secure.

Furthermore, non-compliance allows Quansoft's ability to export the secure PrimeQ run data to Excel for further data analysis. This ability is a key feature in PrimeQ flexibility - 21 CR 11 compliance here would be a disadvantage to our target market - research laboratories. In this way, we try to offer the best blend of practicality and security.

Q. In PrimeQ, can you use individual qPCR tubes and strips?

A. PrimeQ is designed for use with low-profile plates, but single or strip low-profile tubes can be used if they are the same height as a low-profile plate. Many users cut low-profile plates into strips or sections. Either way, it is important to fill the block in each corner to ensure even lid pressure. PrimeQ software can be programmed to read only filled wells.

Q. Can PrimeQ detect a single nucleotide polymorphism?

A. Yes, allelic discrimination is available in the analysis wizard, and can be used for SNP detection.

Q. Can PrimeQ provide a melt point analysis of a plasmid?

A. The sample can be heated in a controlled manner in steps as small as 0.1°C to determine melt temperatures. We recommend initially performing a melt at a bigger increment (0.5°C) then refining this when the Tm has been approximated.

Q. Is the LED light automatically turned on/off during the experiment?

A. The LED is always on when the unit is powered up. The filter wheel acts as a shutter to prevent the light reaching the sample except when performing a reading, at which point the selected filter moves in front of the LED. The PMT is also shuttered. The LED has a very long lifespan.

Q. In an experiment with ROX dye, will the unit automatically subtract the reference dye from the analysis?

A. Yes. Reference dye correction can be turned on or off in the Quansoft Analysis Wizard.

Q. Is the PrimeQ light source a single source across all wells, or is each well individually illuminated?

A. PrimeQ uses a single LED with an X/Y mechanism and mobile fiber optic bundle to travel over the plate for reads.

Q. In PrimeQ do you have the ability to modify temperature ramp rate?

A Yes. Ramp rate can be adjusted in the Quansoft Program Editor.

Q. Is there a glass cleaning protocol for the heated lid to prevent interference with the optical system, affecting the light transmittance?

A. The location of the glass heated lid is such that it does not easily get dirty. However to clean, first ensure the lid is cool then reach inside and gently wipe with a lint-free cloth containing a small amount of 2-butanone.

Q. What is the maximum height for tubes? What would happen if one put in taller tubes?

A. We have tried lots of different low profile plates and strip tubes with seals and flat optical lids and all those tested fit OK. Standard height (non-low-profile) qPCR plates will not allow the drawer to close. If the draw was forced hard, then this would risk breaking the heated lid.

Q. Does PrimeQ software include an efficiency calculator to allow you to determine if the doubling efficiency is close to 100%?


A: A number of factors could be involved in this. Firstly, if the old thermal cycler has not been calibrated recently you may find that there is enough difference in block temperature between the two units to cause the reaction to fail. The reaction has probably been optimized for the original cycler, therefore you may need to look at changing the annealing temperature slightly to adapt it for the new cycler. The ramp rate can also have an effect; changing to a unit with a faster ramp rate may result in less product. You can program the ramp rate of the new unit to match that of the old unit to see if this is having an effect. Finally, the actual temperature profile of the cycler may vary significantly between instruments as they age and especially from one manufacturer to another (i.e. some may over- or under-shoot, some may be set up for predictive tube temperatures, some may take longer to equilibrate to the set temperatures etc). In short, if the PCR is very sensitive you will need to re-optimize the reaction starting with annealing temperature (a gradient can help with this), increasing step hold times and possibly altering the ramp rate.
A. Yes. Quansoft can calculate the quantification cycle (Cq or Ct) by fit points or first derivative maximum. The Cq can then be plotted against the log concentration of the standards used in the experiment to calculate efficiency by slope. A reaction of 100% efficiency will produce a slope value of 3.323.

Q. What is the PrimeQ warranty period?

A. 2 years.

Q. When the drawer is open and qPCR plate inserted, if you push down on the plate, does this risk damaging anything?

A. No, this should not be a problem. The drawer mechanism is strong and the block is sprung.

Q: How can I get copies of the user manuals for the thermal cyclers?

Q. Because PrimeQ has 8 peltiers in the block, does it have the ability to set various temperatures across the 96 wells to optimise primer annealing temperature?

A. No. There is no gradient on the PrimeQ. The usefulness of gradient blocks in qPCR is questionable because qPCR users can just perform a melt (Tm) analysis to determine optimal annealing temp. Tm by qPCR is accurate enough that many labs with a qPCR instrument will optimize PCR protocols on the real-time unit rather than using their end-point thermal cyclers.

Q. Can PrimeQ perform High Resolution Melt (HRM) analysis?

A. PrimeQ Quansoft software can perform melting (Tm) analysis with sufficient precision to distinguish between two oligonucleotides differing by a single base pair, but Quansoft does not contain all the algorithms required for performing High Resolution Melting. However data can be easily exported to Excel to perform the requisite calculations. The block is capable of detecting Tm differences of at least 0.8°C. This procedure is described in Application Note A08-004A.

Q. PrimeQ has a Photomultiplier Tube (PMT) detector, rather than CCD camera. Which is best?

A. A PMT detector is more sensitive than a CCD camera and has a much greater signal to noise ratio. With a CCD detection system there often needs to be a background dye in each well in order to localise the wells for imaging. This is not required with PrimeQ as each well is scanned separately.

A: This would need to be carried out by our service department or by one of our trained service engineers. Please contact service@bibby-scientific.com for further information. All of our calibration equipment is either traceable to UKAS or NIST dependent on the component. UKAS is the UK equivalent of NIST.

Q. Is normalisation software available with PrimeQ, to normalise the expression results from the gene of interest against the housekeeping genes in that experiment?

A. The qPCR software Quansoft allows normalisation to a calibrator sample selected by the user and also calculates the DeltaCq of a housekeeping gene amplified in the same well as the gene of interest. Technical note T08-002A gives further details. There is no instrument normalisation.

Q. Regarding determining copy number, if I need to see/detect 1 single copy, is that possible with PrimeQ?

A. Sensitivity of a qPCR assay is highly dependent on primer efficiency. Not all assays will be capable of detecting a single copy of template in a reaction due to non-specific events. However all qPCR instruments should be able to detect a single copy amplified at 100% efficiency after approximately 35-40 cycles. This is the point at which the copy is amplified to such an extent that it exceeds the detection threshold of the instrument. Techne have demonstrated that PrimeQ can detect a single copy of Lambda DNA (based on quantitation of the DNA using a fluorimetric assay and molecular weight calculations).

Q. Can PrimeQ perform Digital PCR?

A. No, in common with all conventional qPCR instruments, PrimeQ cannot perform digital PCR. This involves a different technology.

Q. Are there guidelines for where to position the unit? For example, should space be left for fans to operate and should direct sunlight be avoided?

A. The manual recommends the following: PrimeQ requires 100mm side clearance for operation. The unit should ideally be operated in the temperature range of 18°C to 30°C out of direct sunlight and draughts. Operating humidity range should be RHa up to a maximum of 80% non-condensing.

Q. Do you recommend that PrimeQ is switched off between runs?

A. No this is not necessary. The unit can be left switched on.

A: User manuals for all current models are available to download from the individual product pages of the website. For older models, please contact
technehelp@bibby-scientific.com

Q. For each of the different analysis options e.g. Arithmetic 1 and 2, Fit points vs First derivative, can you give an indication of advantages/disadvantages and when you might use each of them?

A. Several alternative methods for baseline correction and Cq determination are included in Quansoft in case users are familiar with methods they have used previously. There is little difference between Arithmetic 1 and 2 although when using Arithmetic 1 you need to check that the range of readings selected does not include the start of the amplification.

Fit points is user interactive and so is more flexible for analysing the data and it is possible to alter the parameters to get the best fit. With first derivative, it is based on calculating the Cq from the fastest rate of change of the curve so you have no input. This is likely to be more subject to the efficiency of the reaction but is better for higher throughput analysis. You will receive higher values for Cq using this method so it is not possible to compare experiments analysed by the two different methods.

Q: How can I calibrate my thermal cycler block?

Q. Can the PrimeQ thermal block be changed by the user or do they need a special technician for it?

A. The block is a fixed part of the instrument and should not be removed by the user; it is not intended to be exchangeable. It is best if the block is replaced by a trained engineer as specific tools are required for correct alignment.

Q. How many times does the PCR equipment need servicing in a year?

A. The instrument should not require regular servicing under normal circumstances although the user may wish to have the block calibrated on a regular basis. Block calibration requires special equipment and software but can often be performed by your usual calibration provider.

Q. Pathogen measurements will be performed using PrimeQ. Foil-sealed plates are used for this application. Can a foil-sealed plate be used in PrimeQ, without adversely affecting detection results?

A. It is not possible to use a foil covered plate in PrimeQ as the reading mechanism is through the top of the plate. However, optically clear heat seals or optically clear flat caps are recommended.

Q. Does the design of PrimeQ permit its transportation and use in a mobile laboratory?

A. PrimeQ is not designed to be portable so we would not recommend moving it around excessively.

Q. In calculating Quantification Cycle (Cq) value, what effect does changing Fit Points to First derivative have?

A. The advantage of using first derivative method is that its entirely mathematical and requires no user input. The way in which Quansoft calculates Cq is to take the amplification curve data and calculate rate of change. The fastest point i.e. exponential growth, is taken as the Cq value. When plotted this data gives a peak. The fastest rate of change is the top of the peak. It is important to note that using different methods will shift Cq values so if comparing results from 2 experiments the same method should be used.


Thermal Cycler FAQs


Q: What is a gradient thermal cycler?

A: A gradient thermal cycler allows a temperature step in a protocol to be programmed such that the temperature varies across the block. By specifying a temperature and the gradient to be applied, each column will achieve a different temperature, with the set temperature in the centre of the block, the lowest temperature on the left and the highest on the right. The gradient range is the total difference across the block; for example if the set temperature is 60°C and the gradient 10°C, then the temperatures would range from approximately 55°C to 65°C from left to right.

Q: Why is a temperature gradient required?

A: The annealing temperature of the specific primers used in DNA amplification often requires optimisation. Instead of running multiple experiments with different annealing temperatures the gradient thermal cycler allows the testing of up to 12 different annealing temperatures simultaneously.

Q: Why, according to the gradient calculator, is the gradient not completely linear at each end?

A: The values given by the gradient calculator are the closest to those that will actually be reached in the block. The values at the ends of the gradient will tend not to be exactly the set points programmed by the user due to edge effects. Column 12 will always be slightly cooler due to thermodynamics; without wells on the other side, the edge will experience some heat loss. The temperatures displayed during the run are those programmed by the user; this makes it easier to quickly visualise the programmed gradient. This subject is covered in application note: A01-001A PCR optimisation and gradient.

Q: What is "Hot start"?

A: The Hot Start step is used to pause the machine at a specific temperature, typically around 70°C, after the initial template denaturation. The reason is to allow the manual addition of unmodified Taq DNA polymerase which may loose activity if added during the initial 5min denaturation. Heat-activated Taq or Hot Start enzymes do not require this step.

Q: What is "Sample Cooling"?

A: Sample cooling, where available, can be applied during the lid pre-heat phase. This maintains the block at 4°C while the lid is heating to temperature and avoids any increase in sample temperature due to the heated lid before the thermal cycling program begins.

Q: What is the "Pause" function at the start of the program used for?

A: Some users prefer to preheat the heated lid before placing the samples into the unit. The pause feature is used to stop the unit after the 4 minute heated lid preheat step. It will also sound an audible alarm indicating that the machine is ready for the sample tubes or plate to be added.

Q: What is the incremented time and temperature function used for?

A: Incremented/decremented time and temperature are used to increase or decrease either the time or temperature incrementally over the number of cycles in a stage. Incrementation of extension time is used with "Long PCR" which is when large template fragments are to be amplified (e.g. 27kb lambda DNA, 40kb genomic DNA). Decremented temperature is used for protocols such as "Touchdown PCR" where the first cycle starts with a high annealing temperature and over the number of cycles there is a gradual decrease in the temperature. This ensures that only the specific product is amplified.

Q: What material are the blocks made of?

A: The blocks are made of anodized aluminium alloy.

Q: Can I use adhesive seals to seal my plates?

A: We do not generally recommend the use of adhesive sealing film due to the greater likelihood of sample evaporation. It is advisable, before using expensive reagents, to test the seal. To do this, put 25µl of water in a number of wells, seal the plate and subject it to a typical cycling program. At the end of the run measure the volume using a pipette. A loss of more than 15% indicates sample evaporation and a vapour leak.

Q: What is the smallest sample volume I can run?

A: In the manuals we recommend volumes of between 10 and 50µl. However it may be possible to use smaller volumes if the plate/tube is sealed properly and (where applicable) the silicone rubber mat is used on top of the plate with the lid firmly tightened down. We would recommend a simple test using water samples to see if there is any sample loss. Place a volume of water in the wells, seal and run the program. At the end, centrifuge the plate and measure the sample again using a pipette. For small volumes we would recommend low profile plates sealed using heat seals or caps. The low profile format reduces the amount of condensation on the side wall of the tube, preventing reduction in PCR volume. Low profile products are especially recommended for use with reaction volumes below 20µl.

Q: What is the largest sample volume I can use?

A: The sample in the consumable must be enclosed within the metal part of the heating block for efficient heat transfer. It is likely for larger sample volumes that the hold times for each step will need to be increased to allow the complete sample to reach the set temperature. This may mean re-optimising the reaction for the larger sample volume.

Q: How do I adjust the pressure of the heated lid for my tubes or plates?

A: With the TC-PLUS, no adjustment is necessary. The lid automatically applies the correct pressure. For thermal cyclers with lid adjustment discs, rotate the disc anticlockwise until there is no pressure on the consumable, then close the lid and latch it. To obtain the correct pressure gently rotate the disc clockwise until it is possible to just feel the pressure being applied. Finally, rotate the disc a further quarter of a turn; the lid is now at the correct pressure for use with the consumable. With the 3Prime, no lid adjustment is possible so we recommend the use of dome-capped 0.2ml tubes to give good contact with the lid. For all thermal cyclers, ensure that all consumables used in the block are of the same height and are spread evenly across the block. Insert empty "dummy" tubes if necessary to spread the pressure of the heated lid evenly.

Q: Do I need different blocks for fully skirted and non-skirted 96-well plates?

A: It depends on the model. All 96-well block units in the Prime range will accommodate fully skirted, half-skirted and non-skirted PCR plates. With the TC-5000 and TC-4000 only blocks FTC51BFD and FTC41BFD are suitable for fully skirted plates as they have a slightly taller profile than the standard blocks. They will however also accept non-skirted and half-skirted plates.

Q: How does the TERS® feature of the TC-PLUS work?

A: TERS® harnesses the heat released during the cooling phase for use in the next heating phase. By switching the fans off for a very short time at the end of the cooling period the heat is not removed from the heat sink, but is instead stored as heat energy. Heat is therefore available as soon as the Peltiers switch from cooling to heating. This results in faster heating, requires less energy to heat the block and subsequently costs less to run.

Q: How does auto restart work?

A: Auto restart enables the unit to continue with the run after a power failure. In the case of the TC-PLUS it is possible to set a cut-off time after which the unit will not resume if the power has been off for too long. If the unit was holding at a set temperature when the power was lost, it will go back to that temperature and start the countdown of the step again. If it was ramping, it will continue to ramp to the next temperature. If it was paused, it will go back to the temperature it was at during the pause.

Q: I have a PCR which usually works well but when I tried to run it on a new thermal cycler I did not get such good results. Why?

Q: Is it possible to connect a Prime thermal cycler to a computer?

A: No, not directly. Only TC-PLUS Satellites can be connected to and operated from a PC. However the Techne Workbench PC software allows the user to write programs that can be copied to a USB memory stick and then transferred to the Prime or 3Prime unit. Programs written on the units can also be copied and transferred to Workbench. In addition, Workbench is required to view temperature log files saved on Prime units.

Workbench can be downloaded free of charge from here: Software

Q: Is it possible to connect multiple thermal cyclers to a computer?

A: Yes, up to 9 TC-PLUS satellites can be run either from a TC-PLUS server or directly from a PC using the Techne Workbench software.

Q: How do I upgrade my Prime to a gradient unit?

A: Both 3PrimeX and Prime units can be upgraded to gradient thermal cyclers by purchasing the appropriate upgrade license. For the 3Prime, the part code is 3PRIMEX/USB and for the Prime it is PrimeX/USB.

The license consists of a certificate which contains a unique upgrade code. You will also need the unit serial number and access to the following webpage: Prime upgrade. Follow the instructions given on the license card or user manual to upgrade the unit.