Below is a video I've been trying to publish for a long time coming this summer, but because my summer project only came to a close this week, I've been hesitant to post anything.
In this video, I give an update on my lab work and discuss my overall lab project, which is a great review for myself and my viewers. This is an exciting time for me at Clark University, because I’m beginning the real stages of becoming a graduate student in our accelerated Master’s program. After conducting undergraduate research, I’m using it to support a 5th year of schooling at Clark to finish up my Master’s.
My project concerns the 3’ UTR of the nitrogen-assimilating genes nitrate reductase and nitrite reductase in diatoms (if you haven't read up on my blog before), because we have evidence that along this region these genes are up- and down-regulated by environmental conditions. To test this hypothesis, I’ve created plasmids that drive expression of a reporter gene (GFP) with promoter and terminator regions derived from diatom genes. We’ll compare the activity of GFP with differing 3’ UTR elements to determine whether this region of the nitrogen-assimilating genes is crucial in their regulation.
For the gene nitrate reductase, this is old news. But it is news for my blog that I've completed the plasmids for nitrite reductase, which is really the story of my video.
I'm really excited to be where I am in my project. This week, I hope to genetically transform my diatoms with the plasmids I've created. Oh baby Darwin, this could be a HUGE week for me and my scientific career! I'll be back soon with transformation news.
Showing posts with label 3' UTR. Show all posts
Showing posts with label 3' UTR. Show all posts
Sunday, August 14, 2011
Tuesday, May 31, 2011
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.
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.
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