Fast temperature changes between each step of a PCR program is important for successful PCR reactions. The Eppendorf Mastercycler® gradient thermal cycler provides fast temperature control: up to 3°C per second, by using Peltier elements. It also has the feature of adjustable temperature ramping (1°C to 3°C per second), which gives users more choices when optimizing reactions. The 96-position universal block is the economical option. It can be used for massive amounts of screening work using 96-well plate; single PCR reactions can also be carried out using 0.2 or 0.5 ml PCR tubes without changing the block.
At the beginning of my research work, I started with all kinds of primers. The gradient feature helped a lot. This feature allows 12 different temperatures to be programmed across the block (i.e. each column is a separate temperature). Being able to simultaneously test 12 different temperatures has been very useful when I have needed to test different annealing temperatures for reaction optimization. I only need to choose the range of temperatures; the thermal cycler calculates the exact temperatures for me. This feature is also very useful when I want to do multiple PCR reactions, each with primers using different annealing temperatures. Later, when the cloning work started, the 96-well feature saved me lots of time: Opening/closing and labelling of the individual PCR tubes becomes a pain when screening massive amount of colonies. The 96-well plates made the screening work much easier. The lid of the thermal cycler has three lock positions: one each for 0.5 ml tubes, 0.2 ml tubes and 96-well plates. The lid tightly closes the lids of tubes or covers of plates.
The “pause” command is very useful for hot start PCR. I liked it a lot when I did yeast colony PCR. Because the yeast cell wall is thicker than E. coli, yeast are difficult to lyse. To break open the yeast cells, I started with heating the reaction to 98°C for 3 minutes, followed by 94°C for another 1 min. At this point, the program was paused and polymerase was added into the reactions. In this way, the yeast cells were successfully broken and the polymerase was still fresh when the reaction started.
The thermal cycler comes preprogrammed with standard programs which can be used as templates for more programs. When I started using the thermal cycler, I just modified the lab mate’s program and saved it under another name. The Mastercycler® gradient saves up to 100 programs. All the programs can be printed when the thermal cycler is hooked up to a printer.
If there is any possible drawback, I would say it is that the temperature difference of adjacent rows is not equal when using the gradient feature. For example, when selecting a temperature range of 12°C across the block, we expected the temperature would increase by 1°C in each row. This is not the case and is sometimes a minor inconvenience. However, in summary, I can say the thermal cycler is a user friendly, reliable, good instrument for routine molecular biology work.