I’m back, with results! Today we ran a second gel after re-doing PCR over the weekend. Our gel looked much better, but unfortunately there was still a faint band in the negative control lane, indicating that unfortunately we still had a little bit of bacterial contamination in our sample. We waited to see the results of the other pairs in our group, since each of us ran samples from the other partners, and luckily one group ran a gel with a clean negative control! That means that we were able to move on to the next step of our culture-independent procedure: ligation, which is a way of linking strands of DNA together. Since all of the bacterial DNA from our leaves was all mixed together, we need a way to separate strands from the different types of bacteria to sequence their DNA and determine their species. We’re going to be inserting our different bacterial sequences into plasmids, which are circular strands of DNA found in bacteria. We’ll then insert these plasmids into competent bacterial cells, and use these cells to sequence our original bacterial DNA. We started the ligation process today by preparing our bamboo sample, and will be working on it over the next couple of days.
Simultaneously, we’ve been working on the culture-dependent aspect of this project. Our plated bacteria are growing really well, so today we took colonies of bacteria from the agar plates and made slides out of them so that we can gather data about their shape, arrangement and gram (positive or negative). We did smears of bacteria onto slides, and then stained the slides using a variety of colors (purple, yellow and red—which got everywhere). We had a small panic attack when the decolorizer stain removed all of the sharpie labels from our slides, but luckily we fixed it before we lost three hours of work. These stains will tell us whether our cells are gram positive or gram negative—if it’s gram positive it should stain purple and if it’s gram negative it should stain pink or red. Gram positive or negative gives information about the plasma membrane of our bacteria, which is a first step in classifying their species. Below are some pictures of the slide staining process, enjoy! I’ll keep you updated with what happens when we actually look at the slides this Friday!