Thermal Cycler FAQs

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.

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.

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.

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.

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.

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.

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.

A: The blocks are made of anodized aluminium alloy.

A: We do not generally recommend the use of adhesive sealing film due to the greater likelihood of sample evaporation. However if this is unavoidable then the silicone rubber mat supplied with the TC-4000 and TC-5000 should be used. It is also 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.

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.

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 TC-3000, 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. The TC-PLUS 96-well block 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.

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.

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: 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

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.

A: If only one cycler is going to be connected then an RS232 cable (part code FGEN232) is required. For the TC-3000X and TC-3000G cyclers a USB cable is required (part code 6205046)

A: The table below indicates which power supply, (when there is no TC-512 in the chain), cables and convertor are required for various set-ups. If further assistance is required please contact the Technical Support team at technehelp@bibby-scientific.com