Background and Objectives: Making stacking gels for polyacrylamide gels in the laboratory by conventional methods is laborious and time consuming. Considering the role of temperature in polyacrylamide gels with respect to electrical resistance and viscosity, we assumed that decreasing the temperature would cause an increase in electrical resistance and viscosity. Ultimately, a downward temperature gradient imposed in the first phase of polyacrylamide gel running time would supposedly improve the migration of macromolecules. This project analyzed the effect of temperature gradient on the migration of macromolecules in the continuous gels (without stacking) and compared it with results obtained using stacking gels. Material and Methods: Electrical resistance was calculated using Ohm's law. Subsequently, to examine the effect of temperature change on macromolecules separation, conformation sensitive gel electrophoresis (CSGE) was used as a model and specimens were run under three different conditions, one of which was prepared with a stacking gel. Results: The electrical resistance showed an inverse relationship with temperature in this study. Separation of the DNA molecules in the continuous gels (with no stacking) was comparable with the conventional method (with stacking). Conclusion: Using a temperature gradient against time may be an alternative method for stacking gels.