Taking 1-step intsead of two.

As of late, there have only been a few things on my mind: the glorious weather, Physiological Ecology of Marine Algae, and 1-step Quantitative PCR.

Spring break ended with spring hitting Worcester with extreme velocity. All of the snow that fell before break has been replaced with highs well into the 60s. There are rumors of temperatures hitting the 70s or 80s next week.

Why yes, I'd love some sunshine while I read.

Aside from the end of this week, with temperatures dropping today and rain expected tomorrow, we've been blessed with sunshine and more warm weather. On Monday I got to spend some time outside while doing some reading during class. While we were given more time than needed to do the reading, I certainly will not complain about the amount of sunrays I was able to soak up while hanging outside. I've beginning to worry about the amount of work I have to do between helping to run two projects in PEMA and finishing up my own research, in addition to preparing presentations, writing my thesis, and giving a defense... all while the weather is getting really nice.

I could try shifting my sleep cycle so that I work a lot at night, sleep in, and enjoy the weather during the afternoon before going back into lab. I highly doubt this is something I'll try. Instead, I'll probably start packing killer lunches and having them on the green in the sunshine.

I have so much to do but I'm still waiting for cultures to grow up and things to be mailed in to retry some of my real-time experiments. I tried running some 1-step QPCR reactions recently since we had the kit on hand. 1-step QPCR makes the cDNA and amplifies it all in one reaction, rather than making the cDNA separately and adding it to the QPCR reaction. While this is definitely easier, saves time, and limits contamination, 1-step QPCR is not as accurate when it comes to estimating the amount of starting template in a sample. As determining the amount of relative template among samples of different test conditions is of the utmost importance to me, it looks like 1-step QPCR won't really fit into my plans any more. However, some initial results suggested my experimental lines of diatoms are behaving as expected (and see previously with normal QPCR), so that's really promising. Once I get a new QPCR kit in and some cells to analyze, my work should be all downhill from here (aside from heaps of lab work).

Watch the video below for a bit more on our beautiful weather and 1-step QPCR.

An untitled post about my advisory meeting and a busy semester

Recently, several different groups of people have been asking for permission to use my photos which always is very flattering. Currently, the Clark homepage is using my picture of the Jonas Clark building. Just below that picture on the front page is a video I made, which is a part of a series asking Clarkies what their favorite things are from area restaurants to classes. The Graduate School at Clark has also asked me for my permission to use my photos, but they haven't picked them out yet. Additionally, I've been asked to share my pictures and videos from our October snow storm to be used as part of a new class at Clark that is chronicling the changing landscape of the campus on a historical timescale.

Preparing for my advisory meeting.

At the end of last week (just over a week now), I had my first thesis committee meeting for my master's project. My committee is comprised of my adviser Dr. Robertson, the genetics professor Dr. Thackeray and my undergraduate adviser Dr. Hibbett. The role of my committee is to make sure my project is on the right track, give me advice, and determine whether my work is worthy of a master's degree. This meeting was the first time I've presented my work outside of my lab, so it was a real test of my knowledge and presentation skills. This is something I will be practicing a lot this semester however, since I may be presenting at least thrice more (more about that below). My advisory meeting, save for a few slip ups, went surprisingly well. However, I do have my work cut out for me and it's pretty daunting.

I have the opportunity to present my work at 51st Annual Northeast Algal Society meeting in April, either in a poster or oral format. Additionally, I may also present my work here on campus alongside a collection of research projects representing the Clark Graduate School. These presentations are of course in addition to my thesis defense, which I hope will happen in May.

From my thesis committee meeting and my potentially busy presentation schedule, I have a long list of things I need to accomplish:

• First I need to fix my quantitative PCR reactions, so I can collect data for my project. More on this topic later...
• Second, after I fix my quantitative PCR reactions, I need to complete a lot of bench work that includes growing discrete lines of diatoms, processing them through experimental assays, extracting their RNA, and preparing the RNA for quantification through QPCR.
• This brings me to collecting all of the data I can to support my thesis through many rounds of quantitative PCR.
• In and around here there are multiple experiments and procedures I need to complete to double check on certain things, like sequence my plasmid DNA used to transform the diatoms and check for the number of construct copies each of my diatom lines received in the genetic transformation.
• I also will need to prepare for each presentation and make sure everything is pulled together.

With so many things going on in my research project, I don't think I'll have much time to do anything else. This is of course without considering the work I need to get done in the class I'm helping out with--Physiological Ecology of Marine Algae. It's very likely I will be leading a group research project with 2-4 undergraduate students. This work will be crammed into about 6 short weeks. While I know this will be a great learning experience for me, I know those weeks are going to be very stressful.

New primers and real-time PCR

I've received my new primers and already run a couple different reactions with some results that prove to be promising and others that are frustrating.

Yeah, encouraging :]

The first thing to do with a new set of primers is to run them in a normal PCR reaction to see if the primers amplify the correct length of DNA. While I could do this with my super concentrated, ultra clean plasmid DNA that I used to transform my diatoms (which would serve as the ultimate positive control), I decided to be be bold and try to amplify my complimentary DNA (cDNA) samples. This cDNA was made from mRNA that was extracted from diatom cells exposed to different environmental conditions. Because the mRNA we're looking for contains GFP, anything that is amplified essentially means that our plasmid DNA that we transformed into this line of diatoms is being expressed, which is a great, great thing.

So I ran this PCR reaction with my new primers to amplify my cDNA and this is the gel I got:

There are some pretty convincing bands in that gel which is really encouraging. It appears that all but two of my reactions (6 out of 8) gave us at least some PCR product.

Nicccccce :D

I then ran a real-time PCR reaction called a standard curve, where the source DNA is serially diluted ten fold (I ran 1:1 through 1:10,000 dilutions). For this reaction, I do go ahead and use the plasmid DNA control to give us the cleanest results possible. This reaction allows us to see how efficient the primers are at doubling the amount of DNA product at each PCR cycle.

You can see in this gel (in the first 5 lanes) that a lot of PCR product is being produced. Each reaction hit their saturation point which is why each reaction looks the same even though they had drastically different amounts of starting DNA template. The great this about real-time PCR is that we can see on the computer screen how each reaction was amplified in real time, and see where each reaction it a ceiling amount of DNA. In this reaction I could see that even the 1:10,000 dilution easily hit this ceiling before the 40th (and final) cycle.

With these two encouraging results--the proper amplification using my new primers in a standard PCR reaction and strong amplification in my standard curve using plasmid DNA--I went ahead and tried amplifying my cDNA in a real-time PCR reaction.

Unfortunately, I ran into the same problem I've been having for a few weeks now (see the bottom half of this post).

Sad face real-time PCR :[

Not all of my reactions amplified, and those that did didn't amplify as cleanly as I wanted them to. I ran a gel of my second real-time PCR reaction, which visualizes the two amplified reactions (lanes 3 and 6 starting from the top).

While this is certainly a set back, I have a couple of things I'm going to try. Most importantly, I've ordered a new kit to run the real-time PCR reactions, since the kit I was using was "old." Next, there are a few things I can do to ensure my reactions are as balanced and clean as possible. Finally, I'm going to drop the annealing temperature of my real-time PCR. While I was using the same annealing temperature from my standard PCR, there are two main things that differ between my standard and real-time PCR reactions. First, the salt concentrations are most likely different (although that's a bit annoying to look up, but it's on my to do list), which I found out while screening my diatoms can really screw up a PCR reaction. Second, I used the mysterious "Q-solution" provided by the company Qiagen in their PCR kit when setting up my standard PCR reactions. This may also have significantly changed my standard PCR reaction. While the real-time PCR reaction really should be working with my current set up, it's very possible that my primers are finding it difficult to "seek out" and bind to the sparse cDNA that I want them to amplify.

Marine Biology Research

I made a new video discussing what it's like to do research at Clark. This video pairs really well with the video I made called Bio @ Clark.


Marine biology research at Clark University.