Challenges with Molecular Cloning

Cloning was a major challenge for us. It often took us multiple rounds of digestion/ligation/cloning to get our construct into the cells. Our cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. However, these issues were eventually ironed out and we managed to get the FAP and Spinach into a single construct.
A major help in diagnosing our failures was using the gel consistently to check the length of our inserts, and to sequence periodically to ensure the insert and vector are as expected.
Please refer to our protocols for our final cloning protocol, and remember to reserve some amount of time to allow for experimental failures!


We attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. Using our previous cloning protocols, the cloning using the plasmid vector worked in the first trial. Unfortunately, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols.
The key difference between our plasmid vector and the pSB1C3 vector from the registry was the fact that the submission vector was linearized. We have not been able to pinpoint the exact cause for this, so again, budget sufficient time for cloning!

We attempted to utilize flow cytometry to analyze expression data of our fluorogen-activating biosensors. In order to save time and prevent a queue from forming, we had to fix our cells with 5% formaldehyde before running it in the cytometer. This had negative effects on our results and were unable to see significant signal.