Team:MSOE Milwaukee/Week12


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Week 12


As a group, we took the parts from the kit that we need for the rest of our experiments and transformed them into the DH5 alpha strain of cells. We then plated these cells and are letting them grow for 12 hours overnight. Tomorrow we hope to see colonies on our plates so we can grow a culture in broth for the mini prep procedure. We also ran another gel as planned on Friday, and we did not get clear results. We plan on repeating the gel one more time tomorrow with more DNA in each well to hopefully get more clear bands and quantifiable results.


We checked on the cells in the morning, and the colonies were not yet visible, so we let them grow for a few more hours. We also repeated the gel from Monday, and the bands were still extremely light. We transferred the transformed cells that we grew on plates containing the parts from the kit to broth and let them culture overnight. We didn’t see growth on four of the plates, so we re-plated those four and let them culture overnight.


We completed the mini prep protocol for the plasmids containing the parts from the kit, and we transferred the newly grown colonies to broth to complete the second half of the mini preps. We also ran PCR for the mini prep products of our original 7 genes. After, we ran a gel, and we saw bands in every well that corresponded to the 1,500 mark on the ladder. Finally, we had clear results, even though it meant that something was wrong. Our bands were not supposed to be here, and they certainly weren’t all supposed to be the same. We decided to repeat the gel with the mini prep products before the PCR to find out if the PCR didn’t go as planned. After that gel, we found out that it was not the PCR that went wrong, and we will have to make a plan to test the ligation of our genes into their plasmids. Late at night, we completed the mini prep protocol for the transformed cells that we transferred to the broth in the morning, and this was successful.


We discussed a plan of action for ligating our genes, and we decided to start with the 3A assembly to put together our plasmids and parts for characterization. We decided to start with combining the promoter and the RBS with the standard plasmid so we can use that as a template to insert our genes and terminators for characterization. We also ran a gel from the ligation products to determine in which step there was an error, and we found that the ligation went correctly.


We decided to transform the T7 and RBS ligation into E.coli to continue with our 3A assembly. We also decided to ligate our first five genes to the terminator and pSB1A3 to coninue with our 3A assembly from the other side. These ligations were then transformed and plated. We hope to do another assembly to combine these two parts to start characterization. We also poured LB plates with chloramphenical resistance.