Week 8 - (August 9 - August 13, 2010) My Last Full Week... (sigh)

8/9/10:  My NTS PCR that I did on Friday had been in the fridge for the whole weekend, so I ran it on a gel right away.  I got very peculiar results back - all of my samples were “streaking”, or creating long bands that span the length of the gel.  To be sure it wasn’t the electrophoresis set-up, I cleaned it thoroughly and re-ran the gel, only to get the same reults.  I went even further by throwing out our loading dye (a chemical used to make the PCR samples sink into the wells) and our 1X TAE (used for making the gel and acting as a conductor).  After getting new loading dye and making new 1X TAE I re-ran the gel again, only to have the same results!  Having eliminated the electrophoresis machine as the culprit, I proceeded to re-run the PCR.  Thinking about what I did differently for the previous PCR, I realized I used Matt’s dNTP instead of mine because I ran out.  Matt had similar problems with streaking, so we both concluded it was his dNTP.

Not being able to continue with my research, made more DEPC water, autoclaved (sterilized) beakers and bottles, and cleaned the lab benches as they were getting dirty.  I’m glad I did my lab chores today so that I won’t be in a rush to finish them before my internship ends.

Only seven work days left of my internship, I’m really sad! I wish I could work for another three months.

8/10/10:  I ran a gel on the new NTS PCR that I did and got wonderful results - all of my samples had an NTS signal and my positives worked very nicely.  After talking with Dr. Herfort we decided that I would run another NTS PCR with 1X and 10X dilutions of 18 samples.  Once I run the gel tomorrow, we will choose which samples will be cloned and sent for sequencing.  I’m really happy to get started on the cloning and sequencing.  I just wish I could have been at this point a month ago.  However, due to setbacks in my research this wouldn’t have been possible.

I still have one sample that shows no 18S signal, which we know is impossible because many diatoms and Myrionecta rubra were seen in the sample using a microscope.  Since the sample probably has lots of dissolved organic matter (DOM), I ran another 18S PCR using 1X, 10X, 20X, and 50X dilutions, and doubled the concentration in one PCR tube.  I hope these dilutions work, but I’ll have to wait until tomorrow until I can run the gel.

I met with Dr. Herfort about my poster layout, as this will need to be printed over the weekend.  I found it very easy to start working on since I already made a beginning PowerPoint presentation.  I made a rough layout and I’ll discuss it with Dr. Herfort tomorrow.

8/11/10:  I ran the gel from my NTS PCR yesterday and only one sample had a signal.  This was very odd because we expected at least five of the samples to amplify the NTS region.  My positives also had a faint signal, so I am going to re-run this PCR.  I also ran a gel of the 18S PCR and got no results, even for the positive.  The only difference between this PCR and the others I ran that day was the use of a new dNTP, so I threw this out and got a new working solution of dNTP.

Today I started cloning NH-10 # 906, an NTS sample that had a strong signal on the gel.  By cloning this sample we will be able to send it off for sequencing at the Primate Center in order to identify which variant it is.  Cloning has three main steps:  growing the cells on an agar mixture overnight, growing the cells in a liquid mixture overnight, and a MiniPrep, or preparing the cells for sequencing.  Today I started on the first step, which is growing the cells in a agar mixture on a petri plate.  I already made some plates earlier in July, so I didn’t have to prepare any plates.  Tomorrow, I’ll start the second step of cloning and run more PCRs.

8/12/10:  I ran the gel for the NTS PCR I did yesterday and got no results, yet again.  Since we have been thinking the problem might be the dNTP that I’m using, Vikki gave me some of hers that she knows works.  I re-ran the NTS PCR with Vikki’s new dNTP, and I’ll run it on the gel tomorrow.  If the positive works, but not the sample, there are a few possibilities as to why.  First, it could be that the NTS region of the gene we’re trying to amplify is bigger than we expected, so we’re only amplifying a small part of the NTS region.  Another possibility, which is more likely, is that the DNA has degraded from so much use.  I’ve been using these samples since the beginning of July.  If that’s the case, it will be easy to take a working solution out of the stock of those DNA/RNA extractions.

I also did the second part of cloning today, which involves pricking a white colony of cells from the agar and putting it into the liquid solution.  I prepared eight of the tubes, in case some didn’t work or we needed more DNA for sequencing.  I placed them in a shaker overnight pre-set to 37° Celsius.

8/13/10:  First thing in the morning I had to collect the pellet, or the grown cells, from the liquid medium and freeze them until Monday, when I can do the MiniPrep.  Vikki and Dr. Herfort were out sampling today at Ilwaco Harbor, and I haven’t done the MiniPrep before so we agreed that it would be best to wait until they were back on Monday.

I ran the gel for the NTS PCR and had good and bad results.  The positive now works, so we know the problem was the dNTP.  However, my sample gave no signal, meaning one of two things that I said in yesterday’s post.  To see if it’s just because the sample DNA has degraded I ran a NITS PCR for the two samples that haven’t been working.  If the DNA is still usable, it will give a signal, as this PCR was already done (this is basically a control).  If the DNA is degraded, no signal will be given, meaning I have to make a working solution out of the stock.  If the DNA isn’t degraded, we will have to find different primers for the NTS PCR, since they don’t seem to be working.

I am working on my poster and presentation for the Symposium.  It’s coming along nicely, but I still need to work on it.  I’ll probably have it to the printer by Monday, which makes me a bit sad, because it reminds me that I only have four days left of my internship.

~ Deirdre