Blog #4 Luciferase Assay with TORC mutants

From my last post I mentioned that my CHO reagents no longer worked. For the experiment that I recently just finished, I substituted the CHO reagents with LT1 reagents. As mentioned in my previous posts I maintained my CHO cell line for at least a week before plating them into wells. After plating them into the wells and waiting 24 hours, I transfected my reagents and my plasmids into the cells in the plates. Finally, I performed a luciferase assay after aspirating and lysing the cells to determine their readings.

I tested eight different categories of plasmids which were the LTR, Tax, TORC3, TORC3A, TORC3D, Tax+TORC3A, Tax+TORC3A, and Tax+TORC3D. With these eight categories I receiving my results for 24 tubes. Unfortunately my data did not come out as expected. My TORC3 had an extremely low reading when in previous experiments we had TORC3 having the highest. TORC3D is also supposed to have a lower gene expression than TORC3 but in my results it was shown to be higher. Tax+TORC3 was also unusually low. Tax+TORC3D has higher gene expression than Tax+TORC3. This may indicate a problem during the transfection. TORC3A and Tax+TORC3A seemed to be the only mutant playing its role. It has an extremely high gene expression compared to the others. This mutant is supposed to have a higher gene expression since it is dephosphorylated into the nucleus and is active.

So far my results have not been as expected. This project just shows me how hard research is and how every little thing counts. Especially since my experiments are over a course of three days. Any problems within those three days can greatly alter my results. As for this project, I hope to continue working with Isaacson so I could obtain the ideal results.

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