Sometimes Digests Possess The Answer

Today I'm running a digest on a plasmid in an attempt to cut out a portion of DNA.

This plasmid is the nitrite reductase (NiR) gene terminator region cloned into a vector (called pCR4). Just outside of where the terminator region should be inserted is a series of primer points and restriction sites.  EcoRI restriction sites flank just outside the cloning site, and as such I'm running an EcoRI restriction digest. The digest only takes an hour at 37°C on a heating block (right), and is a simple reaction containing just restriction enzymes, buffer, DNA, and water.

I took a quarter of the restriction digest reaction (5 of the 20µl), added 1µl loading dye, and ran the reaction on a 2% agarose gel:


Here we can see in the second lane my digest. The really bright band is the vector (most of the DNA in the plasmid) and the smaller band (further along on the gel) in my insert, just under 1,000 base pairs. The ladder (the top and 5th lanes) is a 1kb ladder--moving right to left, the smallest band is 500bp, then 1kb, 1.5kb, 2kb, 3kb, and 4kb, etc. I like to use a 1kb ladder most of the time because it's really easy to use, and I can quickly tell which band is which because the 3kb mark is the brightest in the ladder. in my gel here, the vector sequence is larger than 3kb, and roughly equal to the 4kb band (which was expected). The insert that was digested out in the reaction is just short of 1kb (which was also expected.

I then ran the rest of the reaction on a second gel (which was a sort of a waste, but I always want to check my reaction before I look to do anything else with it), because my digest worked. I wanted to cut out the smaller band and use that in a PCR reaction, to make it easier for the primers to amplify them. Since I've been having trouble getting the primers to work on this plasmid, we decided we might as well try this. So I ran the rest of the digest reaction in lanes 2 and 3--you can see where I cut out the bands, which I did with a razor blade (which is the black silhouette on the right).

I then melted the agarose gel that I cut out with my DNA band, and cleaned up the solution. By binding the DNA to a small filter, I could clean the DNA and remove the gel. I then yielded (what I hope is) the digested plasmid insert. I'll use this cleaned up insert from the gel in a PCR reaction overnight tonight and hopefully get some better results.


Below is a cartoon representing the NiR sequence I'm working with. For now, I'm just focusing on the terminator region, which is the 3' untranslated region (UTR) sequence just after the stop codon. By sequencing the DNA we're working with or looking up the desired DNA sequence online at a databank website, we can model the DNA sequence and figure out where the start and stop codons most likely exist. We can then make primers to amplify specific regions along that DNA.

The blue rectangle represents the NiR terminator that we're amplifying: it's just a little bit longer on either side of the actual 3' UTR, which means we're sure to amplify the entire 3' UTR.

More Plasmid Work

I've already talked about how I've made an inducible expression plasmid to test the mRNA stability of nitrate reductase (NR) transcripts in vivo in the diatom Thalassiosira pseudonana. The plasmids run by using a promoter and terminator sequence to run the expression of a reporter gene (we're using GFP). We have a set of NR plasmids, one with a terminal region of NR and one with a terminal region of action (which has nothing to do with nitrogen assimilation). The former plasmid should mimic endogenous activity, whereas the latter plasmid should not mimic the normal conditions in the cell. NR reduces nitrate (NO3-) to nitrite (NO2-), which is then reduced to ammonium (NH4+) by nitrite reductase (NiR). As such, in the scheme of nitrogen assimilation in diatoms, it makes sense to test the regulation of NiR as well.

To do this, I've started work on a set of NiR plasmids. They will also run the inducible expression of GFP, but with their own promoter and terminator regions. Both will have their NiR promoter region, whereas one will have the NiR terminator and one will have the actin terminator region. Both sets of plasmids will be transformed through particle bombardment and then in vivo expression can be measured (through GFP activity).

As it stands, we have a plasmid that contains the NiR promoter & GFP but not the proper terminator. The NiR terminator was cloned into a separate plasmid as part of the process in amplifying out the terminator region of interest. Currently, I'm trying to amplify the terminator out of the plasmid, and in doing so add restriction sites to the plasmid.

The cartoon below represents the series of steps that we have to do in order to manufacture the desired fragment of DNA with restriction sites at the beginning and end of it, which allow us to easily cut out the DNA fragment and place it into a plasmid. Right now, the NiR terminator is represented by the red box. It was amplified out of the entire NiR gene to yield a small piece (between 500bp and 1kb) of the the gene. This piece was at the very end of the open reading frame and extended past the 3' UTR (the terminal region). This was done in a PCR reaction, and the PCR product was inserted into a vector, transformed into bacteria, grown, and then the plasmids were isolated once again the yield the below plasmid.

What I want to do is amplify a smaller portion of the insert out of the plasmid, and make a bunch of copies of it through a PCR reaction. A second set of primers (the green lines) will amplify within the region of the insert, while adding restriction sites. Currently, I've having problems getting this PCR to work because less than half of the primer fits to the DNA of the current insert, while the other half is going to add the restriction site. However, I changed the protocol for the PCR reaction I'm running at the moment. I changed the annealing temperature for the first 10 rounds of my PCR and then I'll bring it back up to what I ran it yesterday (a PCR reaction that did not work, lanes 3-5; right--below right my PCR samples loaded; the faint blue samples are the ladders I used, lanes 1, 2 & 8; the red samples are my PCR reactions that used Coral Load, a special PCR buffer/loading dye combination, lanes 3-7).

...NiR terminator amplification to be continued...

Back to Science

While most of my semester was dominated by finishing up my undergraduate courses, I did get a little bit of work done on my "Monitoring in vivo transcription in the marine diatom Thalassiosira pseudonana using eGFP reporter plasmids" project. Most of my work can be summed up through my post on working on the culture side of the transformational protocol (another post here), setting up media for the transformation protocol, and working on my Academic Spree Day poster and a proposal for my summer work on my 5th year Master's project.

I've had really good success on multiple science projects by putting a lot of effort working on a poster or Powerpoint presentation and then writing my term paper for the project. Below is the final draft of my poster (which I hope to hang up outside of the lab later today), which I presented at Academic Spree Day (ASD).

I had a lot of fun at this year's ASD presenting and sharing my work with science (and non-science) professors and classmates. It was particularly satisfying sharing my poster with fellow science (but non-biology) students, and then going over their posters with them. Certainly a little bit of sharing and learning!

This poster then was the basis for updating my project proposal:

This week is my first week after graduating on Sunday. The name of the game for this week is to get back into the flow of things in lab, and hopefully establish a workable rhythm. At the moment, I'm continuing to work on creating inducible expression plasmids for nitrite reductase.

Graduation: the end of my childhood, and the beginning of being grown up

I graduated! With honors even!

I have to say, even though graduation was much longer than anticipated, it was a lot more fun than I was expecting. Our commencement speaker, Alan Khazei, is a very interesting person and gave an as interesting speech. Mr. Khazei received an honorary degree as did our former university president, our beloved John Bassett. Heaps of pictures from graduation have been posted to Clark's Flickr page, and I encourage you to check them out! I really like a lot of them, and I feel like they accurately display all of the positive energy and emotion of our graduation.

To begin, here are a few pictures of yours truly at graduation:

Just after I was awarded by degree, my friend (who received his accelerated Master's degree) waved at me frantically for a picture as I walked off stage. (His friend took the above picture, however.)

After the ceremony, the campus green was much more of a clusterbunch than I had anticipated, so it was really hard to get pictures with anyone! Here I'm with one of my best friends (Rich), acting all silly like.

I tried my best to recreate one of my first pictures ever on the Clark campus (from Week One)...

...the day I graduated. Props for effort though, right?

It's really hard to imagine that it's already been four great years at Clark! Looking back through my first year pictures on Facebook (while trying to find the above picture) made me realize how fast it's really been. I'm not even halfway done with my combined undergraduate/graduate studies though. From here, it's going to be so much different, but I honestly feel that Clark has prepared me for that. Additionally, by staying a fifth year to complete my Master's, I'll be all set for a Ph.D. program. Interestingly enough, finding a set of Ph.D. programs to suit me is very high on my list of things to do this summer.

Graduation made me realize how much I admire and appreciate Clark for being Clark. So much effort is placed into our mission as a university to educate, inspire, and help the surrounding community that I can honestly be extremely proud to be an alum. I often feel that Clark doesn't get half the recognition it should, but that's not the topic of today's post.

So I passed all of my classes. This means I can graduate!

Let me be clear: in no way was I expecting to fail any of my classes this semester. However, I wasn't expecting to do so well this semester (3.9 GPA). When I got my grades on my iPhone in the middle of an aisle while shopping at Target, I sort of laughed and said, "Well, that's boring" and promptly continued shopping. (I've been shopping around for furniture and the like for my new apartment. More on that later.)

The past month has been very busy--one of the busiest months of my life. I'm still coming down from all of the busting of my butt to get it all done. In due time, I will share everything from finals to papers to posters to graduation (this Sunday, May 22nd, at 1:30PM) to moving into my new apartment and starting my 5th year, all in good time.

I got my cap and gown yesterday (sup, Honors?). My hood, although very complicated-looking (everyone on Facebook was complaining about how odd it was), is quite handsome and I will look like a complete boss on commencement day. I will be sure to have a ton of pictures--a whole photo album worth--from graduation up the following day or so.

Be back soon!

...and it's finals time for Dylan (and everyone else, I suppose)

I have a beautiful, comfortable spot on the second floor of the library, overlooking the gym. Large windows and a lot of natural light, and a very comfortable chair. ^,^