Post 4: Mini Prep Mayhem

Hi All,

To recap – I began the summer by extracting bacterial DNA from 0.22 micron filters that held bacteria from Lake Matoaka. I then amplified the 16s rRNA gene by PCR. These amplicons were ligated into plasmid vectors, transformed into E. coli cells and hundreds of copies of each amplicon were made. What’s next? Since the end goal is to sequence the bacterial 16s gene, I need to get the plasmid back out of the E. coli cell. Enter: Mini-Prep kits. These are brilliant little kits that have made the process of removing plasmid DNA much easier.

To begin, I picked individual colonies into LB broth with kanamycin and grew them up overnight. Once that was completed, I pelleted the bacterial cells out of the broth by centrifugation. (I also archived 500 ul of the culture in 60% glycerol and froze the sample at -20C). Using the reagents in the kit and a benchtop microcentrifuge, I proceded to lyse open the cells and purify out everything but the plasmid DNA. The plasmid DNA was eluted and the plasmid concentration determined by NanoDrop. To date: I have performed 124 mini-preps on the June 2009 Keck Pier samples and 50 mini-preps on the December 2009 Keck Pier samples. Whew!! Click on the link to learn even more about Mini-Preps:

However, this is research. Something had to go wrong! The concentration of plasmid DNA was startlingly low. I was expecting about 200 ng/ul of DNA. I got about 50 ng/ul. I knew there were no contamination issues because my spectrophotometer curves were correct. What could it be? Back to the drawing board. I tried everything I could think of. I increased the incubation time of the broth cultures. I changed the amount of broth I used for the broth cultures. I changed the incubation time of the elution buffer step. I tried to concentrate the DNA in smaller volumes of buffer. I tried different volumes of culture to make the initial bacterial pellet. My friend Zac even did Mini-Preps on two of the samples with his kit. Nothing seemed to work.

However, every cloud has a silver lining. Despite the low plasmid concentration, I still had enough for sequencing reactions. I tentatively handed four of my samples off the Lidia with M13 forward primer. The next day she emailed with great news! The sequences were perfect!! I was able to identify my first four bacterial phyla! It was so cool!!

Now to finish sequencing 44 more June samples and 48 more…