S-STEM Scholar Melanie Castaneda's Blog

  Introduction  During our last week in the lab, we attempted to perform Gibson Assembly with the 3-way fragment we previously assembled and the vector (pRAD1 plasmid). We are also preparing for the summer semester when we will also continue our work with D. metalli .  Methods  5/6/24  We are running out of pRAD1 plasmid extractions. We will need to extract more since we will continue to use it for this project and also for our D. metalli project. We inoculated E. coli pRAD1 into a flask of 25 ml of LB broth. We added ampicillin into the broth before inoculating. Since we plan on extracting within 24 hrs, we placed the flask in the shaking incubator overnight.  We also performed Gibson Assembly. This time we are attempting to assemble the 3-way fragment and the pRAD1 vector. If it assembles correctly, we will have a fragment that is 3,500 kb. We put the 3-way fragment and the vector in a PCR tube with the Gibson mastermix. The tube was incubated at 50℃ f...

  Introduction  Last week we attempted Gibson Assembly for the first time. As we interpreted the results closely, we seem to have been successful. We had a faint band at around 2,500 bp and our fragment was 2,527 bp. Since the band was faint, we are going to attempt to amplify it this week. Methods 4/29/24 We kept our broth culture of D. metalli in the fridge and the biofilm has continued to form. Since it is one of the few species in the Deinococcus genus that forms a biofilm, we were interested in seeing it under the microscope. We decided to do a gram stain on it.  4/30/24 In order to amplify our Gibson product, we decided to run it through PCR again. This time, we did Q5 PCR to get a higher yield than what we previously got. We ran two different samples. The first sample was 48 µL of mastermix + 2 µL of our 3-way Gibson that had been put through PCR last week. The second tube was the original Gibson product that had not been put through PCR.  At the sam...