Team:MSOE Milwaukee/Week9


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Week 9


As a team, we completed the protocol binder and want to double check our work on it. We asked our advisor some final questions on our procedures and determined that we are ready to get into lab. We have a few items left to buy and see if we have them currently in our lab, but we hope to get started in lab late this week, but for sure by early next week.


We started to map out our output plasmids using a free download of ApE software. We had to put the sequences in and it would allow us to label different parts of our plasmid. We hope to use these images on the website once they're completed. We also completed a few miscellaneous tasks.


We signed paperwork to gain lab access and learned more about the labs we will be using. We unpacked boxes of stuff we are allowed to use into the cabinet and got organized. We labeled all of our shelves and took inventory of our stocks. We found out that we didn't have one component for our previously found competent cell protocol, so we found a new protocol online and began day one of that one. We streaked and incubated a LB plate with Dh5-alpha E. coli in hopes of removing a single colony on Thursday.


The competent cell protocol day 2 was carried out in lab. We picked a single colony of Dh-alpha E. coli from our LB plate and placed it into 3 ml of LB broth. The LB broth was incubated and shook at 250 rpm overnight. It seemed to go as planned, but we will not know if it worked for sure until Saturday. We also finished up some safety precaution training for full lab access and we met with iGEM-WLC in order to receive the secretion pump portion for our project. We also found out that our order is coming in sometime on Monday, so we will for sure be able to do more lab work next week.


The competent cell protocol day 3 was carried out in lab. We took 1 ml of our overnight culture and inoculated 100 ml of LB broth for 2.25 hours until it reached an OD600 of 0.383. From this point on, we ensured that the cells stayed cool and we used a MgCl2-CaCl2 and a CaCl2 solution to make our cells chemically competent. After the cells were mixed with the CaCl2, we added 30% glycerol to the E. coli-CaCl2 mixture in order to store the E. coli into the -80C freezer.


In order to determine if our cells were competent, we had to do our transformation efficiency protocol using puc19. One of our potential competent Dh5-alpha E. coli aliquots was used to try to transform puc19 into the cell and also a negative control was used to ensure that ampicilin resistance wasn't already present. Both samples were plated on separate ampicilin plates and incubated overnight.


The competent cell protocol was a success! We checked the growth of the cells, and we had plenty of colonies on the puc19 plate. The other plate had no growth showing that the ampicilin resistance came purely from the puc19 transformation. We plan to use these cells next week once our parts arrive.