Green Fluorestic Transformation Lab
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 minutes.
After 30 minutes, the “- DNA” tube, which only contain the pure E.coli source, will be transferred 250 uL respectively to pure agar media and agar media containing Ampicillin. Meanwhile, the “+DNA” tube, which contains E.coli with fluorescent genes and Ampicillin resistant genes, will be poured 250uL to each media, including containing Ampicillin, and containing both Ampicillin and IPTG. The students will visualize their result in approximately 24-48 hours. …show more content…
This negative result of class may be occurred when the students do not consistently follow the procedure. At the beginning, the student may be lack of twisting the toothpick to inject completely the E.coli colonies to the chloride salt tube, so the amount of E.coli is too small to be selected. In other word, they may add the large amount of salt, which will kill the majority of specimen. In addition, in the transferring step, the student may not suspend effectively, and they transfer unequally to each tube. Another reason that makes the negative result for transformation is that the pFluoroGreen and pFluoroBlue had been added incorrectly to the “-DNA” tube, so the “+DNA” tube, now, contains the normal E.coli, which will not grow in ampicillin plate. The other scenario is that the student had switched the “+DNA” and “-DNA” tubes and poured them in incorrect plate, which would lead them get a negative result for the “+DNA” plate. The low rate of E.coli growth can also potentially occur when the student forgot to add the recovery broth, which helps the bacteria recover their cell wall and membrane faster and avoid to fail to survive. Furthermore, the “heat shocked” cycle may not be performed enough time for the cell to form the pores on their cell wall to allow the potential exogenous DNA to enter the potential