Temperature and Time Optimization of pGEM-T Plasmid Transformation in Escherichia coli JM109

Abstract

Introduction: The widely used method for transformation was the heat shock method, which was influenced by the time and temperature of heat shocktreatment. However,therewasalackofresearch on the optimal temperature for pGEM-T plasmid transformation in Escherichia coliJM109. This study uses the heat shock method to determine the optimum temperature and time for pGEM-T transformation in Escherichia coli JM109. Methods: The research method used was a quasy experimental. The researched unit was the plasmid pGEM-TandEscherichiacoliJM109competentcells, with six treatment groups with temperature variations of 35°C and 42°C, for 30, 60, and 90 seconds and a controlled group that contained DNA insert. The transformed plasmids were cultured and then isolated to assess the intensity of the plasmid bands on agarose gel electrophoresis using ImageJ analysis. Results: The results of the band width measurement obtained the largest value of 44. 874-3.380 pixels in the 42°C- 60-second treatment group. At the same time, the results of measuring the intensity of the band obtained the highest value of 2.2235 in the 42°C-60-second treatment group. Conclusion: The results indicate that the optimum condition for pGEM-T transformation in Escherichia coli JM109 was at 42°C for 60 seconds.