by Brennan, Josh, and Grace
Introduction:
After being sent home back in March due to COVID19, we are finally back in the lab continuing with our research. The biggest change since we started research again is that our group is now a group of three rather than two separate groups. We first started off with a warm-up lab to allow us to become comfortable with lab itself. We still meet every week as a lab group to discuss what we did in lab, how it turned out, and if there was any way to improve out protocols if we needed to. Now that we are halfway through the semester, we are quickly approaching the end of our research for the school year. We have been able to achieve a lot since coming back to campus when it comes to lab, and we will continue to move forward with protocols while making sure we are following safety guidelines in regard to COVID19.
Research:
The past two weeks we have been doing plasmid purification and restriction digest protocols for our miRNAs. During plasmid isolation our main goals were to grow overnight cultures and to prepare plasmid DNA by miniprep. In the plasmid isolation protocol, we picked colonies from plates containing our miRNA bacteria and added them to 24 tubes, 8 for each one of our 3 miRNAs. After the bacteria were added to the tubes, they were allowed to grow out overnight. The next day all of the tubes had growth and were cloudy. After the tubes had growth, we moved onto the next step which was preparing the plasmid DNA by miniprep. During this step we used a kit containing various solutions that would allow us to purify our plasmid DNA. For this step, we chose to purify the two tubes for each miRNA that had the largest DNA pellets. Once those tubes were purified, we Nano dropped them and got the concentration of plasmid DNA for each miRNA. We began screening clones by restriction digest after we found the concentration of each sample. The first step of the restriction digest was to choose tubes that had concentrations closest to 50 ng/ul (three of our samples did). Once we had tubes with high enough DNA concentrations, we made an individual master mix for each tube. The master mix was created, and we began to make a gel, so we could see if our plasmid had the insert from miniprep. After we ran our gel, we analyzed it and wrote the results in our lab books.
Throughout the protocols, we stumbled a little bit. We were producing great results up until we had to find the concentration of our samples. With only three of our samples having a high enough concentration, we felt a little bit behind. However, we were still able to run a gel. We anticipate needing to run another gel due to the fact the bands came out curved and overall looked incorrect. Once we are able to finish another gel, if we are able to, we will send any successful miRNA’s to be sequenced this week. As we are approaching the end of the semester rather quickly than we anticipated, we are hoping to continue with our research and produce better results for when we present our information.
Reflection:
The results for plasmid purification and restriction digest were not necessarily what we would have ideally liked to see. For many of our tubes produced in plasmid purification the DNA pellets were rather small and would yield small concentrations. All of this aside we still managed to have some tubes that produced respectable DNA concentrations. For our restriction digest we had only one sample out of three that didn’t work. This means that we will need to go back to previous steps to get better DNA concentrations as we expect them to be more likely to work.
