S-STEM Scholars Sam Bennett's Blog

    This week we started with running a couple gels for our different fragments. I worked on the left fragment, which unfortunately did not give the results we were hoping for. Since it was all the same sample, just ran on different temperatures in the thermal-cycler, we combined them all and will work later on cleaning them up.     We took our e.coli broth and used that as our base. Originally we started with only 1mL of our sample, and after the plasmid extraction was completed, there was very little DNA in the sample. We then used 5mL to extract enough DNA to work with. We started with centrifuging 1mL at a time and discarding the supernate. After we did 5 different cycles of centrifuging we had our pellet     Once we had the pellet we resuspended it with 200 micro-liters of our B1 buffer. Then added an additional 200 micro-liters of the B2 buffer (Lysis Buffer), followed by gently inverting the sample until it was mixed. An additional 400 micro-lit...

 Introduction     This week we hit the ground running. We started by taking our fragments of DNA and started to put them together. We took 3 different samples, two being DNA and the other being our plasmid. Inside of the samples we had 1 micro-liter of our forward primer, 1 micro-liter of our reverse primer, 2 micro-liters of the DNA/Plasmid, and finally 16 micro-liters of master mix. Once all the samples were combined, they were placed inside the thermal cycler to run their course. Upon completion of the thermal-cycler, we came to find out there was a mix-up in which DNA/Plasmid was placed into our tetramycin sample. Since we took the time to create the samples and had them run in the thermal cycler, we decided we would run the sample on the nano-drop as well as run a gel.      As seen from the gel there are a couple ghost fragments as well as blurriness, we'll be working towards cleaning the sample for the cleanest fragments we can get.    ...

 Introduction     Thursday was spent preparing bacteria for whenever the supplies we need arrive. We started by dyeing some samples with Crystal Violet in hopes of it sticking to biomass. After the samples had grown for 48 hours, we evacuated the walls (emptying the trays) and then rinsed them with water, then dumped them out again. After the samples had been successfully evacuated, it was time to apply the dye. We placed 225 micro-liters of Crystal Violet into the samples, allowed them to sit at room temperature for 20 minutes, then repeated the evacuation steps. After the dish has been successfully evacuated, we allow the samples to rest for 24 hours at room temperature being placed at an angle to dry.      Come Tuesday, our samples will hopefully reflect the growth of biomass, and we will continue the process of inserting a new fragment into our sample's DNA.