Good news! The last two days in lab have been really successful, and we’re finally getting to the point where we’re close to results. On Tuesday evening, I went into lab and prepared our two culture-independent samples for Qiagen column miniprep, which we did in lab yesterday. This is a multi-step process that purifies our plasmid DNA. After purifying our samples, we ran a restriction enzyme digest. The enzymes cut our DNA in specific places, so tonight I ran a gel to estimate the size of the cut pieces and to make sure our bacterial DNA inserted correctly. Our gel looked really good, and the bands were in the correct place, so tomorrow we can send our two samples off for sequencing! I’m not sure how long it’ll take to sequence them, but hopefully by sometime next week we’ll get results and can start comparing our sequences to a database that will (hopefully) allow us to determine the species of bacteria present in our phyllosphere.
Yesterday, along with doing the Qiagen column miniprep, we re-ran a gel of our culture-dependent PCR products, because last time our negative control lane was contaminated. Luckily, when we re-ran the gel our negative control lane came back negative, so we were able to proceed with this portion of the project. What was really exciting was that all but one of our samples came back positive for bacterial DNA, so, assuming everything works from here on out, we’re going to have a lot of culture-dependent data! At this point, we need to run basically the same procedures on our culture-dependent samples as we did on the culture-independent samples. Yesterday we performed a ligation on our samples, and tonight we went into lab and transformed them. Part of the transformation process involves spreading two agar plates per sample, so I ended up spreading 34 plates! Our samples are currently taking up half the incubator, but by tomorrow, if everything goes well, we’ll be able to do pre-plasmid purification prep on our samples, run a Qiagen miniprep and restriction enzyme digest on them on Saturday, and run a gel on Sunday. If everything looks good, we’ll be able to send our samples out to be sequenced on Monday! I have my fingers crossed that everything goes well in the next few days, especially since we’re getting so close to the end of the quarter (2 weeks!) that it’s time to start thinking about what we’re going to put on our poster that we’ll be presenting the last week of class.
When we had some down time yesterday we collected more bamboo leaves (with stems) for future staining. Something interesting I learned about bamboo is that it has flowers! Professor Wilson showed us that at the base of the leaf, where it connects to the stem, are small hair-like structures, which are specifically designed for wind pollination. We also learned that, although bamboo isn’t full of pectin (sugars), it might actually be a good habitat for bacteria, which would explain why we have so many different samples. Even though the leaves don’t have a lot of nutrient sources, they also don’t produce anything that’s deleterious to bacteria, unlike daffodils, which apparently produce a substance that’s toxic to most types of bacteria! I included some pictures of our bamboo flowers and one of our pre-plasmid purification preparation plates from Tuesday.