Trouble with culturing and amplification (Week 3)

This week's work was cut short by the 4th of July, but it feels like I got a lot done. The majority of my time this week was spent on extracting DNA from 9 more soil samples. It took me a little longer than necessary to complete the extractions, because I didn't want to work with too many of the samples at once, so I did it in two batches. I wasn't too familar with the technique yet, so I wanted go more slowly and make sure I didn't make any mistakes.

After extracting DNA from the first set of samples, I attempted to amplify the DNA with PCR. But when I ran the samples on a gel, it was clear that the PCR  hadn't worked. I did the same thing with the next set of DNA and this time I got better results. Some of the DNA amplified and some of it didn't. This left me with the question of why some samples were amplifying strongly and some were not. Rick and I discussed two possiblities: either the DNA had some left-over ethanol from the extractions that was keeping it from amplifying, or some of the caves had such a low biomass of bacteria that there was minimal DNA in the sample. The ethanol problem could be fixed by evaporating all the liquid from the DNA and then re-suspending them. So that's what I did. Hopefully, the heat from the evaporation didn't denature the DNA. First thing next week I plan on doing a PCR on the DNA from all the samples to see if they will amplify now.

In addition to extracting DNA, I also began some more cultures this week. I was disappointed to find that the plates I had streaked last week did not grow any bacteria. So, I started a new set of plates, using more of the sample than previously. I am putting some of the plates in the 10 degree room and some of the plates I am leaving at room temperature. It's likely that the room temperature ones will grow fastest, but maybe the bacteria will surprise us.

In my spare time between culturing bacteria and extracting DNA, I learned how to make media and how to make a DNA loading buffer. We needed a different type of media to grow Rick's deep-sea bacteria, so I learned how to make SWC media and then I poured it into plates. SWC media is a rich media which uses real sea-water. Next week I plan on transfering the bacteria to the SWC plates, in hopes that it will glow! Also, I had to make some more loading buffer. I have run so many DNA samples on gels in the last couple of weeks that I used up the all the loading buffer. I made a 10 mL batch. Rick says that will probably last for another 2 years, so I won't have to worry about running out of it again this summer.