Week Four: NSF visit and technique improvement

What a busy week!  I spent most of my time this week working on my PCR and nitrate quantification technique but this week was also the NSF visit.  What a wonderful group of scientists to have around to pick their brains!

I guess we'll start with Monday.  If you remember, on Friday of last week, I ran a PCR to amplify bacterial amoA genes.  When we looked at the results on Monday, it was pretty much a mess.  My standard curve was all over the place, and I had multiple melting points which is bad.  So on Monday I tried again just with the standards (no samples) to try and tighten up my standard curve.  Because were working with such small volumes (1µl sometimes), even the smallest variation in volume transferred is obvious.  I was extra careful about watching my transferred volumes and when the PCR was finished, my hard work paid off.  My standard curve had a correlation of 1 (!) but my slope was a little bit off.  This suggests that I was perfectly precise in my volume transfers (every transfer was exactly equal), but they weren't exactly accurate which threw off the slope of the curve.  Also, one of my controls showed some amplification around 35 cycles, but this is probably the result of non specific binding so we won't worry about it.

On Tuesday, we autoclaved pipette tips, microcentrifuge tubes and other lab necessities.  We also made up our standards for nitrate/nitrite analysis.  This was done by a colorimetric assay kit by Cayman Chemical Company.  Basically, the sample is added to the wells, and then converted to nitrate.  The nitrate is then converted to an azo product which gives it its purple hue.  The darker the sample, the more nitrate in the sample.  We can also measure the nitrite concentration by skipping the conversion to nitrate step.So, in this image, we have our standards on the left two columns, increasing in concentration down each row so that a standard curve can be calculated.  The four brightest wells in the middle are the samples that have all be converted to nitrate, and the four wells below those are measuring only nitrite.  Pretty cool stuff.

On Wednesday, we prepared the medium for our sediment samples that will be used to incubate them.  This is kind of a lengthy process, but take away message is this is the solution we put our samples into.  I also extracted DNA from soil like last week, and Jeff came in and took my picture for the webpage.  Very exciting!

Thursday was the NSF visit.  The day started with a general overview of CMOP and the things we do here, presented by director Antonio Baptista.  Following that were two presentations about the first two initiatives, ocean acidification and plankton blooms respectively.  For lunch I had the privilege to sit with Dr. Spence and Dr. Van Dover, two very intelligent and friendly women.  They asked us about our research and our plans for the future and gave some good advice and shared some personal experiences with us.  After that I got some stuff done in lab, and measured the nitrate/nitrite levels for our first two days.  We also measured ammonia concentrations from the water we collected from our samples by measuring the absorbance in cuvets. 

That brings us to Friday.  What a busy Friday indeed.  We extracted RNA from our day 0 sediment.  My goodness is that an involved process that takes forever!  We extracted our RNA but we still need to purify it this week.  We also extracted our day 3 water sample that we can measure our nitrogen compounds from and measured the nitrite/nitrate concentrations.  This time we diluted our samples because they are already reaching the upper limits of our standard curve.

Anyways, that's definitely enough for now.  This week should be another busy one.  We're going out in the field this week so I'll write all about that soon!