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.

I personally have been having some issues preparing one of the plasmids that I need to transfect into cells. While doing preps of other plasmids, we have been getting acceptable concentrations that we can use in experiments. For my plasmid, Ha-Ub we have been getting extremely low yields. After talking to Dr. Hinton, we decided to order a new bacteria to try and increase our yield, but so far it does not seem to helping at all. I’m going to continue trying to increase the yield for this plasmid,so I am continuing to try and troubleshoot, just like I have been for the whole summer so far.

Comments

  1. mnwatson says:

    Altering protocols seems to be a theme for the summer – my lab partner and I have been having similar struggles with getting the right volume ratios of our different reagents to get better RNA yields. I guess it’s part of research and experimentation, but its definitely disheartening to keep trying and to keep getting disappointing results, especially when protocols are time consuming, but stick with it! I’ve got my fingers crossed for better nucleic acid yields for all of us 🙂

  2. aikashmanian says:

    I’ve also had to redo some parts of my project because we couldn’t get our spectrometer to work properly! Research really is a lot of troubleshooting. I’ve learned a lot from observing how graduate students and PIs in my lab attack problems like these, and it seems like you’re in problem solving bootcamp. As I’m sure you’ve learned, it’s so important for scientists to not get discouraged after some part of the experiment fails. I think that’s one of the most important lessons I’ve learned from this summer experience. I hope you were able to solve the problems you have been having and that everything is going smoothly now!