User:Derek K.L. Chu/Notebook/Investigation training
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Transformation: Investigate the best recovery time of ampicilin, kanamycin, chloramphenicol, etc in our lab.
Transformation using antibiotics as a selection system requires a recovery time for the cell to express antibiotic resistance gene. This takes from 30 to 60 minutes. Our group is trying to investigate the minimal recovery time needed for a successful transformation, in order to save the time for cloning.
We define the best recovery time as the shortest time needed to get a usable amount of colony. We assign 6 colony as the amount of colony needed for a convincing transformation result. The project is planned to measure the colony forming unit (CFU) for transformation using different antibiotics. The project is planned to test for the CFU using 5 different recovery time. Transformation using ampicillin (AMP), kanamycin (KAN) or chloramphenicol (CHL) as a selection system for transformant are tested. In our project, we use pCMV/myc/mito/GFP, pEGFP-N1, pSB1C3-BBa_K1119003 for the transformation using AMP, KAN, CHL as the selection system for successful transformant. We use DH10B as the competent cell.
We first search for the amount of DNA needed for transformation which would give a countable CFU. After selecting the amount of DNA for giving CFU within countable range, transformation using different recovery time using the chosen DNA amount was carried to search for the best recovery time.
For the first part of experiment, we transform the cell with different amount of DNA, using 1 hour of recovery time. From previous measurement, the transformation efficiency of the competent cell is 1*10^8 CFU/ug. To generate plate that give CFU within range of 30~300CFU, 1pg, 10pg, 100pg of DNA was used for testing the amount of DNA needed.
1. Transformation using different amount of DNA
The data for AMP and CHL are not conclusive. As it is shown above, there are transformation with the result of replica with large discrepancy. Since we failed to maintain the confidence of our experimental result, we decided that none of the data can be used to support a conclusive analysis.
For the KAN transformation, cell lawn was obtained for all recovery time. In fact, it was recalled that our KAN plates did not pass the plate testing. For the negative control test, there were bacteria grown on a DH10B plate. Therefore, we were unable to collect any data from the KAN.
Since the result of the replica does not agree with each other, we are unable to draw any conclusion based on the data.
Statistical software eg Minitab could be used to calculate the correlation coefficient between the recovery time and the CFU. The p-value will tell the significance of the correlation, and the sign of r will indicate whether it is positively or negatively correlated.
Appendix: Protocol for transformation
transformation using different amount of DNA
7) Centrifuge the cell at 13.2krpm for 30 seconds, remove 900ul supernatant, resuspend the cell pellet by gently pipetting it.