“A molecular mechanism for Alzheimer’s disease: the effects of WT and mutant Presenilin D257A on TrpC5 channel function” Blog #2

Summer research at Pace in the biology labs with Dr. Buraei has been an incredible learning experience. Right away we began by preparing competent cells by growing E.Coli overnight, then utilizing ZymoBroth and buffers to obtain the highest efficiency cells as possible.
During our first trial using the Zymo Mix & Go kit, we followed each procedure exactly as directed. The largest concern was making sure that each step was carried out at the correct temperature. As a majority of the procedure required conditions which were 0-4 degrees Celsius, we made sure to have a container of ice on hand at all times, and precooled the equipment needed. This is crucial since a cells competence can decrease immensely when it reaches temperatures higher than 4 degrees Celsius. Additionally, once prepared, the cells must be stored at -80 degrees Celsius, so in order to prepare we labeled the micro centrifuge tubes ahead of time as well as cooled them using ice.
Although our first trail was successful, after further research we learned the competency can be greatly increased by using a few troubleshooting techniques. For our research, we want to use cells with the highest transformation efficiency as possible, meaning the cell has the best chance of accepting the foreign genetic sequence including our desired mutation. Two of the main challenges in the second trail were growing the E.Coli culture in the ZymoBroth at room temperature, as opposed to 37 degrees Celsius, and being even more precise with keeping everything at a low temperature once the bacteria grew to our desired level. Since bacteria will optimally grow at 37 degrees Celsius, lowering the temperature will result in a much longer doubling time, taking a greater amount of time to reach our desired optical density. However, once grown, we decided that in order to further reduce the temperature of the culture to rise we could keep all tubes and pipettes which we would use in a freezer, in addition to labeling the micro centrifuge tubes and placing them in the -80 degree Celsius prior to use. Unfortunately, we found that we had allowed the overnight culture to grow in the ZymoBroth for an extended amount of time and it had passed the optimal optical density. However, since the cells were still usable for a different step in our research, we were able to quickly finish that trial and begin again.
In our last trial making the cells we followed the same procedure as the second trial, this time adjusted the calculated doubling time and monitoring the culture every two hours. This adjustment, along with preparing the tubes and pipettes before hand resulted in very successful competent cells which will be crucial in our next steps in research!
Over the next few months I am thrilled to continue this research with Dr. Buraei and to perform electrophysiological studies to hopefully draw a sound conclusion!

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