User:Stephanie P T. Yiu/Notebook/Restriction Enzyme Activities at 4°C, 37°C and 65°C

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Method

For this experiment, pSB1C3-BBa_K516030 plasmid obtained from iGEM Kit Plate 2013 was selected as the digestion assay sample. 1 μl of the sample was transformed into E. coli, inoculated and allowed to grow overnight. The plasmids was extracted from E. coli and purified using alkaline lysis miniprep. One positive experimental setup and one negative control were prepared for each reaction environment, i.e. 4°C, 37°C and 65°C.

Both positive and negative setups contained 300 ng of pSB1C3-BBa_K516030. XbaI and PstI-HF were used in digestion and were not added in the negative control. Instead, it was compensated by an additional 0.4 μl ddH2O to keep the total digestion volume at 18 μl in accordance to protocol. One set of both positive setup and negative control was placed in refrigerator at 4°C. The other two sets were incubated at 37°C and 65°C respectively.

After 1 hour of digestion, gel electrophoresis was carried out to inspect the digestion result.

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