More about primers (Week 6)

Most of this week was spent working with primers. Firstly, I had to optimize the new primer sets. I used the qPCR machine, so that I could do the samples at different temperatures all at one time. This was the biggest PCR I'd ever done: 44 tubes. I can't imagine filling up an entire 96-well plate. After that, I ran all of the PCR results on gels. The results weren't as clear as I had hoped. (That seems to be a major theme in scientific research: none of your results are as clear as you would like.) But it seems that 60 degrees Celsius is a good hybridization temperature for all three primer sets.

Unfortunately, these new primers are forming lots of "primer dimers" during the  PCR. That's when the primers attach to eachother instead of the DNA in the sample. The fungal primers are especially troublesome in this respect.

This week Rick started to look at how many barcoded primers we will need to buy for the sequencing. Once we get these primers, I can use them to amplify and barcode each of the samples. Then, we will send them to be sequenced. After that will be the most exciting part. I will be able to figure out which bacteria are in each of the caves! It's hard to believe that this is already the end of six weeks and I only have four more weeks of research. There seems like a lot of work left to do in only four weeks, but Rick says there is plenty of time.