Our Final Week

With coming to a close in the summer, things are winding down in the lab. Many of us have gotten incredibly exciting results, and look forward to getting more results over the school year. I have prepared all of my experiments, I will just have to continue working to visualize the results over the school year. I am certainly looking forward to continuing to work towards a publication. For our last week, our lab helped to throw a barbecue for the biology department students and professors, as a celebration for everyone’s hard work. The whole lab went grocery shopping, prepared for the bbq, and cooked together, which made for a very nice close to the summer. We all had a wonderful time together this summer, and I am looking forward to continuing to work with everyone during the school year!

Getting Good Bands

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Western Blotting Woes

For the past few weeks, I’ve been working diligently to try and prepare lysates for my experiments. With most of my lysates finished, I prepared to move into western blotting for visualization. At the NCI, I performed many western blots, so I was fairly confident in my ability to get good bands. During the school year, I also tried to visualize some proteins, to no avail. I was hopeful to get better results over the summer, but we are still trying to troubleshoot. We’ve been trying to determine if the antibodies are working properly, and to see whether or not the transfer was good. Using a stain called ponceau, we found that our first transfer method was not actually transferring the protein onto our membrane. Because of this, we switched to a semi-dry transfer method. We think that we have figured out what the problem is, so this week we are looking forward to getting nice bands on our film!

A Success in Trials

After dealing with some plasmid troubles for a few weeks, I’ve finally found some success! After changing bacterial types, plasmid preparation kits, and eventually the DNA that I was transforming from, I was able to get a decent yield. In the end, I had to use DNA from a different tube, one that I had already prepped during the school year. I was originally trying to transform from our original tube, but I have a feeling that the concentration in that tube was just too low for the plasmid to copy well in bacteria. During the plasmid prep that I got to work, I still got a fairly low yield, but it is certainly workable. Luckily, I only need to transfect in about 200 ng of plasmid per well, so as long as my DNA is at least 100 ng I can use it.

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Week 4: Troubleshooting

This week, we’ve been having some issues with our plasmid expressions, both when transfecting in cells, and in trying to prep them for purification. All of us in the lab have been having issues with some of our plasmids, so the other veteran members and I have been trying to figure out what the problem may be. For expressing in cells, we decided that we may need to alter our transfection protocol, because the DNA may be too toxic to express at such high levels. Talking with one of the other members, we decided to vary the amount of our transfection reagent, as well as the levels of DNA. It seems that we may have figured out how to alter our protocol, but we are going to have to do some more experiments to continue our optimization.

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