It's the last day of the quarter. Thank god for that.
Things haven't been going well recently with the cloning. We've been having difficulty in most of the steps so far, sans the step where we introduce the insert into the bacterial plasmid. Plates failed to grow when all indications showed they would thrive, PCRs have turned up negative despite huge amounts of DNA and now someone transferred our plates to the wrong incubator without telling us, shaving a day off.
Now, one last assay has emerged on the horizon. How much of each kind of bacteria are there in the soil? After we standardize the DNA concentration between the different regions, we can finally get an estimate of just how different things are in the varying locations along the river. Using PCR, we will use all 8 different primer sets, and enhance the present DNA to a point where we can show the abundance of difference families of bacteria well.
Ok. Back to spanish. Big test tomorrow morning.
Friday, June 20, 2008
Sunday, May 25, 2008
Snags
So, as of now we haven't gotten very far at all into the cloning process...the first steps of mixing the DNA and plasmid and getting them into the bacteria worked, but only just. When we went to plate our solutions out to pick the white colonies from the blue colonies, we got bad results, which either manifested as a lawn of bacteria, or a small amount of blue and white colonies, in tight, hard-to-work-with groupings. According to Bill, however, everything we were doing seemed right on the money, so he's currently incubating another batch so we can look at them this coming tuesday.
Hopefully, it will give us lots (50+) distinct colonies per plate, and not an unusable lawn or 'hot spots'. Once that is over we can pick and patch and grow each colony properly by itself to isolate the certain DNA sequence.
Hopefully, it will give us lots (50+) distinct colonies per plate, and not an unusable lawn or 'hot spots'. Once that is over we can pick and patch and grow each colony properly by itself to isolate the certain DNA sequence.
Friday, May 23, 2008
Presentation
I am surprised, honestly, at how well our section of the Elwha presentation went at the Olympic National Park visitor center. The presentation itself felt way too short and simple compared to everyone else's, but somehow it all came together into a sweet and succinct description of what we had been doing over the past year. Thanks to Jessica, I had someone to play off of and to keep my head on straight throughout the entire presentation, but as soon as we hit the questions, that's when it got easy.
Details were always easier to remember for me than being asked to create my own summary of things...
At any rate, like I said in the presentation, cloning has started. We just mixed the plasmid and the DNA fragments together, then introduced them into the E. Coli cells day before yesterday. Hopefully, by today, we should have some good colonies to pick and patch, and things can go briskly before Bill needs to leave.
Details were always easier to remember for me than being asked to create my own summary of things...
At any rate, like I said in the presentation, cloning has started. We just mixed the plasmid and the DNA fragments together, then introduced them into the E. Coli cells day before yesterday. Hopefully, by today, we should have some good colonies to pick and patch, and things can go briskly before Bill needs to leave.
Monday, April 28, 2008
Fat man in a footrace
Now that the massive string of PCRs and gels are finished, we're just starting on Denaturing Gradient Gel Electrophoresis. We used some of Bill's samples today, from a few other projects that he has been working on. The gels themselves are quite thin compared to the normal and vertical, rather than horizontal, and have their own difficulties, mainly in the loading process.
DGGE works on the basis of the actual base pair sequence in certain pieces of DNA being different from the others. For instance, in strands which contain lots of Guanine and Cytosine bonds, it will begin to denature, or 'melt' at a higher temperature than ones which contain more Adenine and Thymine. This is due to the fact that the G-C unit in DNA has 3 hydrogen bonds, while the A-T unit only has two. Since the DNA molecule overall has a negative charge, it will tend to be drawn down towards the bottom of the gel, due to electrical fields being applied.
The whole concept of gel electrophoresis is that these negatively charged DNA fragments, varying in length from anywhere between 250 to 1500 base pairs long, will move through the gel 'mesh' towards the positive electrode on one end of the apparatus. But just like a fat man in a foot race, the larger and larger pieces will fall behind progressively and form their own 'bands' in the gel, which is then visualized under UV light to confirm it's presence.
What DGGE does however, is that it has a chemical which, at just the right concentration, will cause these pieces of DNA to split apart, but only halfway. Imagine a very rudimentary ladder as a DNA analogue, but then cut it apart down the middle and splay the two ends out in a Y formation. Suddenly, the DNA fragment can no longer move through the gel, as it is now snagged on the molecular matrix. It's' sorta like walking through a crowd, but when someone gives you a signal you have to open your arms wide. Suddenly, you can no longer move nearly as quickly towards your goal.
Where they get stuck in the gel tells us a lot about it's overall sequence makeup, including how to differentiate between different mutations of the same region of DNA sequence. For instance, the technique is being used in cancer research to see whether or not certain mutated sequences of DNA are correlated with cancer. In the cell biology and microbiology realm, it has been used to differentiate between notoriously mutation-prone mycobaterium.
In our work, we're planning to use it to look for the number of different sequence mutations in our DNA samples, which gives us a big clue into how many different species of bacteria reside in the soil.
DGGE works on the basis of the actual base pair sequence in certain pieces of DNA being different from the others. For instance, in strands which contain lots of Guanine and Cytosine bonds, it will begin to denature, or 'melt' at a higher temperature than ones which contain more Adenine and Thymine. This is due to the fact that the G-C unit in DNA has 3 hydrogen bonds, while the A-T unit only has two. Since the DNA molecule overall has a negative charge, it will tend to be drawn down towards the bottom of the gel, due to electrical fields being applied.
The whole concept of gel electrophoresis is that these negatively charged DNA fragments, varying in length from anywhere between 250 to 1500 base pairs long, will move through the gel 'mesh' towards the positive electrode on one end of the apparatus. But just like a fat man in a foot race, the larger and larger pieces will fall behind progressively and form their own 'bands' in the gel, which is then visualized under UV light to confirm it's presence.
What DGGE does however, is that it has a chemical which, at just the right concentration, will cause these pieces of DNA to split apart, but only halfway. Imagine a very rudimentary ladder as a DNA analogue, but then cut it apart down the middle and splay the two ends out in a Y formation. Suddenly, the DNA fragment can no longer move through the gel, as it is now snagged on the molecular matrix. It's' sorta like walking through a crowd, but when someone gives you a signal you have to open your arms wide. Suddenly, you can no longer move nearly as quickly towards your goal.
Where they get stuck in the gel tells us a lot about it's overall sequence makeup, including how to differentiate between different mutations of the same region of DNA sequence. For instance, the technique is being used in cancer research to see whether or not certain mutated sequences of DNA are correlated with cancer. In the cell biology and microbiology realm, it has been used to differentiate between notoriously mutation-prone mycobaterium.
In our work, we're planning to use it to look for the number of different sequence mutations in our DNA samples, which gives us a big clue into how many different species of bacteria reside in the soil.
Thursday, April 24, 2008
Mass Production
Finally, the work I've been waiting for all year has come! PCR, Gels, cloning and DGGE. No more nasty soil samples to work with or mindless stick-soil-in-tube-wait-half-hour protocols. By friday evening, 6 more PCRs and their respective gels will be complete, followed closely by Denaturing Gradient Gel Electrophoresis and some cloning into E. coli bacteria.
The DGGE and cloning are going to be the ways that we can discover exactly what is in the soil. For now, there's just six tubes filled with a LOT of different bacterial DNA, and no way to draw any conclusions about their respective regions. By isolating and splicing the DNA into a bacterial genome, we can create a clone colony which represents the DNA's former owner.
There'll be more details in the next post
The DGGE and cloning are going to be the ways that we can discover exactly what is in the soil. For now, there's just six tubes filled with a LOT of different bacterial DNA, and no way to draw any conclusions about their respective regions. By isolating and splicing the DNA into a bacterial genome, we can create a clone colony which represents the DNA's former owner.
There'll be more details in the next post
Thursday, March 13, 2008
Primers are here
This is great news! Once the respiration portion is finished, hopefully by tomorrow, we can finally begin working on the bacterial DNA. Now we have all the equipment to find out what microbes we have in the soil.
The primers are pieces of DNA oglionuclueotides (don't worry, I didn't know what they were either), which basically tell the DNA polymerase in the PCR machine what pieces of DNA it should replicate and amplify.
1. Heat to nearly 100 C denatures the DNA by breaking the bonds holding the double strands together, and the DNA unravels.
2. The DNA is cooled to about 60 C and the primers latch on to their corresponding sequences in the denatured DNA.
3. The taq polymerase (DNA synthesizing protein found in hot springs bacteria) attaches to these bonded strands and rebuilds them into two strands. The temperature here is about 75-80 C
4. Repeat until the DNA has been sufficiently amplified. The reason behind the bacterial polymerase is that it can withstand the temperature cycling required to break the DNA strands apart. Normal human polymerase would break down at such high temperatures.
http://en.wikipedia.org/wiki/Polymerase_chain_reaction
Can't wait for it!
The primers are pieces of DNA oglionuclueotides (don't worry, I didn't know what they were either), which basically tell the DNA polymerase in the PCR machine what pieces of DNA it should replicate and amplify.
1. Heat to nearly 100 C denatures the DNA by breaking the bonds holding the double strands together, and the DNA unravels.
2. The DNA is cooled to about 60 C and the primers latch on to their corresponding sequences in the denatured DNA.
3. The taq polymerase (DNA synthesizing protein found in hot springs bacteria) attaches to these bonded strands and rebuilds them into two strands. The temperature here is about 75-80 C
4. Repeat until the DNA has been sufficiently amplified. The reason behind the bacterial polymerase is that it can withstand the temperature cycling required to break the DNA strands apart. Normal human polymerase would break down at such high temperatures.
http://en.wikipedia.org/wiki/Polymerase_chain_reaction
Can't wait for it!
Saturday, March 1, 2008
Out of Time
I can't believe I only have 2 posts for this quarter. I could have sworn I did 3 already. Grr.
Since I last posted things have gone to hell in a handbasket, and are now holding steady in the 'up **** creek' level. Spanish is done for. Even after agreeing to take an incomplete this quarter I still kept falling behind, so I'm going to have to audit and try again later. My grades have slipped down to 'average' in Calculus, and now, with less than a month to go, we're finally getting somewhere in Chem.
Now I'm screwed, cause drafts for all three REU assignments are due on Monday, and I have no idea even where to start. The fact that I'm the only person officially in my group doesn't help. And that the work that I'm doing now can only be described as mind numbingly boring.
The experiment is to measure the respiration rate of the soil and compare it to the amount of organic microbial carbon present in the soil, in order to draw conclusions about how the microbial activity varies based on the conditions above, below, and between the dams. It also provides a baseline of data to allow comparison in future studies.
There, just summarized the whole quarter in 4 lines. That's really interesting and all, but I don't really feel that Mr Joe Average cares. Animals and fish, maybe, cause he likes hunting his healthy animals and fish, but I doubt he is interested in how those N-fixing bacteria are doing. If we took a vidcast of the last few days of work, the most interesting thing would probably be me scooting around the room in a chair at high speeds to alleviate boredom. That is, unless staring at a computer screen for 4 hours straight is more fun.
A research brief, on the other hand, I can actually do. I can attempt to hook an high school audience with the work I'm doing, but since they're all either pregnant or on drugs these days and could care less and my brief will probably end up buried god knows where, I'll go for you guys. It won't be easy reading, but I don't expect the final journal article to be any better.
I really don't like how much of these last few posts have become so negative, but The last quarter has made me grumpy and bitter, and an outlet like this is hard to come by. I apologize once more.
In other news, the main experiment for the microbial group is on the path to completion. The whole chair racing thing I touched on earlier was about the last step, measuring the respiration rate of the soil. It really is mind-numbing stuff. 1. Hydrate the soil 2. Wait an hour 3. Put the soil in the device and hydrate another sample 4. Wait 30 minutes 5. Take the soil out and reset the tube for 15 minutes. 6. Repeat steps 3 - 5 until all 28 samples are done.
Once that is all done though, we'll have all the data we need to complete the experiment and relate the respiration to the microbial carbon. After that, it's all DNA work, which will hopefully be far more sexy in the public eyes.
So when I come to class on Monday, in all likelihood I'll only have my research brief, with maybe a podcast script if I have some sort of brainwave. I'm sorry Dwight, but I don't like BSing to anyone about this stuff, and I really don't think that what we've done so far is nearly interesting enough for a podcast or vidcast. Even you'll be bored to death. There's a reason only one other person applied to the Microbiology group last quarter.
Since I last posted things have gone to hell in a handbasket, and are now holding steady in the 'up **** creek' level. Spanish is done for. Even after agreeing to take an incomplete this quarter I still kept falling behind, so I'm going to have to audit and try again later. My grades have slipped down to 'average' in Calculus, and now, with less than a month to go, we're finally getting somewhere in Chem.
Now I'm screwed, cause drafts for all three REU assignments are due on Monday, and I have no idea even where to start. The fact that I'm the only person officially in my group doesn't help. And that the work that I'm doing now can only be described as mind numbingly boring.
The experiment is to measure the respiration rate of the soil and compare it to the amount of organic microbial carbon present in the soil, in order to draw conclusions about how the microbial activity varies based on the conditions above, below, and between the dams. It also provides a baseline of data to allow comparison in future studies.
There, just summarized the whole quarter in 4 lines. That's really interesting and all, but I don't really feel that Mr Joe Average cares. Animals and fish, maybe, cause he likes hunting his healthy animals and fish, but I doubt he is interested in how those N-fixing bacteria are doing. If we took a vidcast of the last few days of work, the most interesting thing would probably be me scooting around the room in a chair at high speeds to alleviate boredom. That is, unless staring at a computer screen for 4 hours straight is more fun.
A research brief, on the other hand, I can actually do. I can attempt to hook an high school audience with the work I'm doing, but since they're all either pregnant or on drugs these days and could care less and my brief will probably end up buried god knows where, I'll go for you guys. It won't be easy reading, but I don't expect the final journal article to be any better.
I really don't like how much of these last few posts have become so negative, but The last quarter has made me grumpy and bitter, and an outlet like this is hard to come by. I apologize once more.
In other news, the main experiment for the microbial group is on the path to completion. The whole chair racing thing I touched on earlier was about the last step, measuring the respiration rate of the soil. It really is mind-numbing stuff. 1. Hydrate the soil 2. Wait an hour 3. Put the soil in the device and hydrate another sample 4. Wait 30 minutes 5. Take the soil out and reset the tube for 15 minutes. 6. Repeat steps 3 - 5 until all 28 samples are done.
Once that is all done though, we'll have all the data we need to complete the experiment and relate the respiration to the microbial carbon. After that, it's all DNA work, which will hopefully be far more sexy in the public eyes.
So when I come to class on Monday, in all likelihood I'll only have my research brief, with maybe a podcast script if I have some sort of brainwave. I'm sorry Dwight, but I don't like BSing to anyone about this stuff, and I really don't think that what we've done so far is nearly interesting enough for a podcast or vidcast. Even you'll be bored to death. There's a reason only one other person applied to the Microbiology group last quarter.
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