Green Fluorescent protein and Green Fluorescent protein are suggested to be involved in bacterial transformation tool, which helps the scientists visualize the normal protein in cell. To test the hypothesis, the experiment had been collected the around 15 colonies from E.coli source plates. Those amounts of E.coli, then, were used to test under transformation of E.coli with Green and Blue Fluorescent Proteins procedure. As the result, the E.coli that had been injected pFluoroGreen and pFluoroBlue will be survived in the Ampicillin environment, and they can express the fluorescent characteristic supporting by the IPTG, which helps to release the RNA polymerase (T7). The results also shows that the transformation efficiency rate of the class
…show more content…
Around 15 well-isolated colonies will be picked up by a toothpick, and transferred to the same tube that contains CaCl2. This tube will be suspended until no clumps of cells are visible and the cell suspension looks cloudy. 250 uL of “- DNA” will be transferred to “+ DNA”. The “+DNA” tube will be added 5 uL of each pFluoroGreen and pFluoroBlue. Two tube of E.coli, now, will enter to the “heat shocked” cycle. At first, they will be incubated in ice for 10 minutes, before dipped into the 42C water bath for 90 seconds. Immediately, students will return those tube to incubate in ice for 2 more minutes. In order to help the cell wall and membrane of E.coli recover quickly, 250 uL of recover broth will be added to each tube, before those tubes, again, are incubated in 37C water bath for 30 …show more content…
Moreover, the student should twist the toothpick more to drop down more the amount of E.coli. In the suspending step, the specimen should be stayed in the machine long enough until the cloudy solution appears, so the amount of E.coli will be separated equally to the “+DNA” and “- DNA” tube. The other issue should be improved is that the microbiology lab should offer more suspend machines for student due to the sensitive time consuming of the experiment. In particular, while the student is waiting for their turn in the machine, the chloride salt may over lyses the cell wall of the E.coli bacteria and kills the majority of them, which will affect significantly to the rate of transformation
Around 15 well-isolated colonies will be picked up by a toothpick, and transferred to the same tube that contains CaCl2. This tube will be suspended until no clumps of cells are visible and the cell suspension looks cloudy. 250 uL of “- DNA” will be transferred to “+ DNA”. The “+DNA” tube will be added 5 uL of each pFluoroGreen and pFluoroBlue. Two tube of E.coli, now, will enter to the “heat shocked” cycle. At first, they will be incubated in ice for 10 minutes, before dipped into the 42C water bath for 90 seconds. Immediately, students will return those tube to incubate in ice for 2 more minutes. In order to help the cell wall and membrane of E.coli recover quickly, 250 uL of recover broth will be added to each tube, before those tubes, again, are incubated in 37C water bath for 30 …show more content…
Moreover, the student should twist the toothpick more to drop down more the amount of E.coli. In the suspending step, the specimen should be stayed in the machine long enough until the cloudy solution appears, so the amount of E.coli will be separated equally to the “+DNA” and “- DNA” tube. The other issue should be improved is that the microbiology lab should offer more suspend machines for student due to the sensitive time consuming of the experiment. In particular, while the student is waiting for their turn in the machine, the chloride salt may over lyses the cell wall of the E.coli bacteria and kills the majority of them, which will affect significantly to the rate of transformation