Optimize Reactions in a Single Experiment
Molecular biology laboratories routinely need to optimize incubation temperatures for thermal cycling reactions. Optimization is critical but not always easy to do. Even after calculating the Tm (melting temperature) of a primer, the annealing temperature often needs to be determined empirically. This involves repeating a reaction at many different temperatures. Similar time-consuming tests may also be required to optimize the denaturation temperature.
The thermal gradient feature on the 96-well block of the DNA Engine multi-bay cyclers allows you to optimize assays in a single experiment by using a range of temperatures simultaneously. At any step in a protocol, a temperature gradient of up to 25°C may be programmed across the reaction block.
Predictable and Reproducible Gradient Temperatures
The temperature gradients formed by Bio-Rad thermal cyclers take a nonlinear, yet highly reproducible shape that allows accurate prediction of the actual sample temperatures. Extensive validation of the gradient temperatures on multiple reaction blocks indicates that the nonlinear gradient is both predictable and reproducible.
Temperature values displayed for each of the 12 columns of the reaction module on a DNA Engine cycler. Traces plotting measured temperature from four cyclers have been superposed, showing the cycler-to-cycler consistency. Note that the size of each point is far smaller than the specified error (±0.4°C).
Precision (Dynamic) Ramping Ensures That the Time Spent at the Incubation Temperature Is the Same for Each Sample
Precision (dynamic) temperature ramping means that the temperature gradient is formed during ramping. This ensures that each sample well reaches set temperatures (including gradient and uniform set temperatures) at the same time, and thus that the incubation period is consistent across all samples in the experiment. This is critical for objective evaluation of the temperature and assay conditions.
Dynamic ramping in gradient mode. Four thermal cyclers were programmed to develop a 45–65°C gradient across 12 columns. Thermal measurements were taken with NIST-traceable, laser-trimmed thermistors from 48 wells (four wells/column) in each cycler. Mean temperature of each column in each cycler (48 total traces) is plotted. Note that the software adjusts ramp rates so that all samples reach the incubation temperature at the same time.
Gradient Calculator
The gradient calculator for DNA Engine thermal cyclers tells you the incubation temperature of each column.
Programming a gradient step lets you determine the optimal temperature for a specific assay or test a range of annealing or denaturation temperatures in a single thermal cycler run.
This virtual gradient calculator uses the same algorithm that predicts well temperatures for a gradient step in DNA Engine thermal cyclers. Enter the lowest temperature (>30°C) and the highest temperature (<105°C) you want to use, and the calculator will indicate the incubation temperature that will be used for each column. The temperature accuracy (±0.4°C) ensures that at the end of a single optimization protocol you will know the optimal temperature for your experiment.
Bio-Rad thermal cyclers use dynamic ramping, which means all wells reach their target temperature at the same time, so all samples stay at target for the same duration. Therefore, both the incubation time and temperature can be easily transferred from your optimization experiment to your standard protocol.
Note: Gradient is available when the cycler is outfitted with a 96-well reaction module.
Virtual Gradient Calculator
