I've been attempting to read a lot of papers in the past week, and struggling fairly badly. Reading primary literature is hard for two reasons: you need to have enough background knowledge to understand the experiments, and you need practice at parsing the language and form of papers (a nontrivial skill). I've come a long way since high school, when it took me several days to look at all the words in a paper (and I still came out the other end with little understanding of what actually happened)... but I still have an awfully long way to go.
Which is why I love review papers. The literature search is done, the information is chewed-but-not-digested. It's just the right level for an undergrad. (Book chapters can also be good, but seem to be a bit less up to date because the publication cycle is longer.) A review paper is the best way to start a new literature search, or to get the "state of the union" on an interesting area you're not expert in.
<3 people who write review papers
Friday, August 7, 2009
Saturday, August 1, 2009
The strawberries are delicious
Today I decided to try a DIY DNA extraction. I started with Meredith Patterson's informal instructions, figured out what chemistry was actually happening, looked up some protocols from actual labs, and found what might be the original paper presenting the method of "salting out" DNA. Perhaps more research than I really needed to do, but it was pretty satisfying.
Then I went out and acquired a bunch of things: non-iodized salt, isopropanol, ethanol, papain (aka meat tenderizer), some "test tubes", a "centrifuge"... and strawberries.
For some reason, strawberries seem to be the canonical thing to extract DNA from if you're just demonstrating, so you can show your audience slimy white strands and have them go "ooh, aah". Maybe they have a lot of DNA or something. (What ploidy are generic commercial strawberries?? EDIT: they are apparently octoploid. Impressive!)
Right now, one mashed-up strawberry is sitting in a solution of salt (I should probably be using a real buffer), shampoo (disrupts cell membranes and precipitates proteins), and papain (slices up proteins). All the amounts of things were totally guessed. I put the solution in a double ziploc bag and set it on top of a hot computer case to speed up the reaction. This is the first step, where I break apart the tissue and lyse the cells to get at the DNA. I'll leave the strawberry to digest overnight, and tomorrow I'll centrifuge to try and remove as much protein and random crap as possible, then add concentrated salt and isopropanol to precipitate the DNA. If all goes well, I might even post photos.
Watching the strawberry get slowly digested is interesting by itself. The red juicy parts got digested first, turning the solution a lovely pink, and leaving behind all the white fibrous stuff from the center. Did you know each seed on the outside of a strawberry has a tract of white-fibrous-stuff connecting it to the center of the berry? Since all the red stuff surrounding those tracts has already been digested, it's a little like looking at the skeleton of a strawberry. Kind of creepy. We'll see what happens by tomorrow morning.
...Oh, a word about test tubes and centrifuges. For test tubes, I went to a florist and bought some of the plastic tubes they use to keep single flowers moist. Ten cents each, and with neat little lids. For a centrifuge, I was toying with the idea of modifying a blender or something, but then I found the lid to a salad spinner, which lots of internet people have used as a makeshift centrifuge. Unfortunately, not the whole salad spinner, which would have been brilliant; but I added some zip-ties and it became a centrifuge capable of generating about 32 Gs. This is probably good enough for a proof-of-principle DNA isolation (though of course I want to do more than prove the principle). So far, I haven't spent any money on lab equipment that's sold for the purpose of being lab equipment. I'm going to have to get fancy sometime soon, because you can't do bacterial work with a piddling little 32-G centrifuge; you need something that can get up above 10,000 Gs. (Ebay, here I come!)
Then I went out and acquired a bunch of things: non-iodized salt, isopropanol, ethanol, papain (aka meat tenderizer), some "test tubes", a "centrifuge"... and strawberries.
For some reason, strawberries seem to be the canonical thing to extract DNA from if you're just demonstrating, so you can show your audience slimy white strands and have them go "ooh, aah". Maybe they have a lot of DNA or something. (What ploidy are generic commercial strawberries?? EDIT: they are apparently octoploid. Impressive!)
Right now, one mashed-up strawberry is sitting in a solution of salt (I should probably be using a real buffer), shampoo (disrupts cell membranes and precipitates proteins), and papain (slices up proteins). All the amounts of things were totally guessed. I put the solution in a double ziploc bag and set it on top of a hot computer case to speed up the reaction. This is the first step, where I break apart the tissue and lyse the cells to get at the DNA. I'll leave the strawberry to digest overnight, and tomorrow I'll centrifuge to try and remove as much protein and random crap as possible, then add concentrated salt and isopropanol to precipitate the DNA. If all goes well, I might even post photos.
Watching the strawberry get slowly digested is interesting by itself. The red juicy parts got digested first, turning the solution a lovely pink, and leaving behind all the white fibrous stuff from the center. Did you know each seed on the outside of a strawberry has a tract of white-fibrous-stuff connecting it to the center of the berry? Since all the red stuff surrounding those tracts has already been digested, it's a little like looking at the skeleton of a strawberry. Kind of creepy. We'll see what happens by tomorrow morning.
...Oh, a word about test tubes and centrifuges. For test tubes, I went to a florist and bought some of the plastic tubes they use to keep single flowers moist. Ten cents each, and with neat little lids. For a centrifuge, I was toying with the idea of modifying a blender or something, but then I found the lid to a salad spinner, which lots of internet people have used as a makeshift centrifuge. Unfortunately, not the whole salad spinner, which would have been brilliant; but I added some zip-ties and it became a centrifuge capable of generating about 32 Gs. This is probably good enough for a proof-of-principle DNA isolation (though of course I want to do more than prove the principle). So far, I haven't spent any money on lab equipment that's sold for the purpose of being lab equipment. I'm going to have to get fancy sometime soon, because you can't do bacterial work with a piddling little 32-G centrifuge; you need something that can get up above 10,000 Gs. (Ebay, here I come!)
Friday, July 31, 2009
Bitesize Bio is shiny!
I just discovered Bitesize Bio, an awesome site full of discussions of common molecular-bio lab questions and problems. It has an RSS feed but is also set up with categories and menus for non-blog-style browsing. The ~5 posts I've read have all been informative, well-written, and interesting. I got a couple about interesting new techniques, a couple about theoretical questions and their practical consequences, and a couple about being a good grad student or a good mentor. It's really shiny, especially for a new lab member like me who wants to know the reasoning behind the magical incantations we sometimes do. "Wash with 0.75mL Buffer PE"? What does Buffer PE even do? ...OK, that's a cheating example -- Buffer PE is a proprietary mix from a commercial kit (but I'm told that after you add ethanol to it, as you must, it's just an improvement on straight ethanol).
For example, I just read an article about touchdown PCR, a neat hack on traditional PCR.
Sometimes in a PCR reaction you'll have trouble with the primers binding in incorrect places, because there happens to be some random sequence in your sample that's sorta-kinda complementary to your primers. Then you get nonspecific amplification of random crap, which can drown the gene fragment you actually wanted to amplify. What do you do?
You can calculate the optimal annealing/melting temperature (Tm) of your primers, and make sure to do your annealing step at that temperature. But random salts and stuff in your reaction can affect the Tm, so the calculated Tm is only an approximation. And at any temperature reasonably close to the Tm, even if it's not optimum, some annealing will happen. Maybe not much, but some. That's thermodynamics for you.
The idea of touchdown PCR is that there's a sweet-spot temperature where, statistically, it's too hot for nonspecific annealing, but just cool enough that the correct annealing can happen. You can't know exactly where this temperature is -- and you wouldn't want to run your whole reaction at that temperature anyway, because you'd only get a small amount of primer annealing and your yield would be low. So instead, for your first cycles you use an annealing temperature significantly higher than the calculated Tm for your primers (about 10 C higher). Then, in subsequent cycles, you gradually lower the annealing temperature. So in early cycles, only a few primers will anneal, and they'll almost all anneal to your actual sequence of interest, and not to random other sequences that are close-but-a-little-off. By the end of the cycle, you've amplified your correct sequence by a little bit relative to incorrect sequences. Run through several more cycles, and by the time you get to a "standard" annealing temperature where nonspecific annealing can happen, you've hopefully amplified the correct sequence quite a bit already, so nonspecific annealing becomes less of a problem because there are just more copies of the correct sequence available for the primers to bind to.
When I read about that, I was blown over. It's such a clever thermodynamics hack! -- it takes advantage of the fact that chemical reactions (DNA base-pairing or anything else) have fuzzy, stochastic behavior. For a given reaction, there isn't a sharp cutoff temperature where it goes from "too hot to react" suddenly to "OK now reaction proceeds fully". It's fuzzy. If it's too hot, a few molecules will react. Get a bit closer to the optimal temperature, and more molecules go. If you have two competing reactions with slightly different optimal temperatures (like specific and nonspecific annealing!), it's like having two bell curves overlapping, centered at slightly different values. You can find a value where one bell curve is acceptably high and the other is very low -- and then once you've amplified your chosen sequence N-fold, the game changes and you don't have to worry about the incorrect sequences nearly as much. It's like magic!
References (from original post):
1. Roux KH. Genome Research. 1995. 4: S185-194.
2. Mattick JS et al. Nature Protocols. 2008. 3(9). 1452 - 1456
For example, I just read an article about touchdown PCR, a neat hack on traditional PCR.
Sometimes in a PCR reaction you'll have trouble with the primers binding in incorrect places, because there happens to be some random sequence in your sample that's sorta-kinda complementary to your primers. Then you get nonspecific amplification of random crap, which can drown the gene fragment you actually wanted to amplify. What do you do?
You can calculate the optimal annealing/melting temperature (Tm) of your primers, and make sure to do your annealing step at that temperature. But random salts and stuff in your reaction can affect the Tm, so the calculated Tm is only an approximation. And at any temperature reasonably close to the Tm, even if it's not optimum, some annealing will happen. Maybe not much, but some. That's thermodynamics for you.
The idea of touchdown PCR is that there's a sweet-spot temperature where, statistically, it's too hot for nonspecific annealing, but just cool enough that the correct annealing can happen. You can't know exactly where this temperature is -- and you wouldn't want to run your whole reaction at that temperature anyway, because you'd only get a small amount of primer annealing and your yield would be low. So instead, for your first cycles you use an annealing temperature significantly higher than the calculated Tm for your primers (about 10 C higher). Then, in subsequent cycles, you gradually lower the annealing temperature. So in early cycles, only a few primers will anneal, and they'll almost all anneal to your actual sequence of interest, and not to random other sequences that are close-but-a-little-off. By the end of the cycle, you've amplified your correct sequence by a little bit relative to incorrect sequences. Run through several more cycles, and by the time you get to a "standard" annealing temperature where nonspecific annealing can happen, you've hopefully amplified the correct sequence quite a bit already, so nonspecific annealing becomes less of a problem because there are just more copies of the correct sequence available for the primers to bind to.
When I read about that, I was blown over. It's such a clever thermodynamics hack! -- it takes advantage of the fact that chemical reactions (DNA base-pairing or anything else) have fuzzy, stochastic behavior. For a given reaction, there isn't a sharp cutoff temperature where it goes from "too hot to react" suddenly to "OK now reaction proceeds fully". It's fuzzy. If it's too hot, a few molecules will react. Get a bit closer to the optimal temperature, and more molecules go. If you have two competing reactions with slightly different optimal temperatures (like specific and nonspecific annealing!), it's like having two bell curves overlapping, centered at slightly different values. You can find a value where one bell curve is acceptably high and the other is very low -- and then once you've amplified your chosen sequence N-fold, the game changes and you don't have to worry about the incorrect sequences nearly as much. It's like magic!
References (from original post):
1. Roux KH. Genome Research. 1995. 4: S185-194.
2. Mattick JS et al. Nature Protocols. 2008. 3(9). 1452 - 1456
Thursday, July 30, 2009
I return
Hello all,
Yes, I've been away from this blog for a very long time. But I find myself having more science content that I want to talk about, so I figure it's time to revive the Dendritic Arbor.
(Originally, I tied this blog not to my main Gmail account, but a different one. This turned out to be really annoying because every time I signed in to Blogger, I got signed out of my email. This was a major disincentive to post, and since I didn't realize you could transfer blog ownership between accounts, I just posted less and less. But now everything is fixed. Hooray.)
Where am I now? Well, I'm between my sophomore and junior years at MIT, I've radically changed my social circle, and I've pledged a (coed) fraternity, Epsilon Theta. More relevantly, I've finally entered a lab where I'm doing productive work on an interesting project in an environment I like, after a couple of short and ill-fated other lab positions. I'm working in one of the wellsprings of synthetic biology, Tom Knight's lab, studying a very intriguing and not very well-known bacterium.
At the same time, I'm also trying to get more into DIY Bio -- cheap open source lab equipment, electrophoresis gels in drinking straws, that sort of thing. We'll see how this goes. I may or may not be too busy to ever get started on this, what with classes (those random annoying peripheral activities that colleges insist you participate in).
It's good to be back.
Yes, I've been away from this blog for a very long time. But I find myself having more science content that I want to talk about, so I figure it's time to revive the Dendritic Arbor.
(Originally, I tied this blog not to my main Gmail account, but a different one. This turned out to be really annoying because every time I signed in to Blogger, I got signed out of my email. This was a major disincentive to post, and since I didn't realize you could transfer blog ownership between accounts, I just posted less and less. But now everything is fixed. Hooray.)
Where am I now? Well, I'm between my sophomore and junior years at MIT, I've radically changed my social circle, and I've pledged a (coed) fraternity, Epsilon Theta. More relevantly, I've finally entered a lab where I'm doing productive work on an interesting project in an environment I like, after a couple of short and ill-fated other lab positions. I'm working in one of the wellsprings of synthetic biology, Tom Knight's lab, studying a very intriguing and not very well-known bacterium.
At the same time, I'm also trying to get more into DIY Bio -- cheap open source lab equipment, electrophoresis gels in drinking straws, that sort of thing. We'll see how this goes. I may or may not be too busy to ever get started on this, what with classes (those random annoying peripheral activities that colleges insist you participate in).
It's good to be back.
Monday, September 29, 2008
Open Courseware is not the be-all and end-all
MIT's Open Courseware gets a lot of press. And a lot of it is well deserved -- it's fairly comprehensive, and does a good job managing the technical side of throwing vast amounts of material up on the internets (hooray for PDF). But it's not everything.
The past few days I've been stressing over various topics from my genetics class, mostly yeast tetrad analysis. Going to lecture hardly helps; for this class, it's been generally true that the more confusing topics are very poorly explained. Usually the textbook does a good job of explaining things, but it doesn't cover yeast tetrads hardly at all. And I have a test in two days.
(Yeast tetrad analysis is pretty interesting, by the way. I might write a post about it if I ever actually understand it.)
Turning to OCW didn't help, because more or less the same professors have been teaching the class for N years, so the lecture notes are exactly the same. I can get practice problems (old homeworks and exams), which are very useful once I actually understand something, but not in this case.
So I turned to Google. And lo and behold, googling {yeast tetrad analysis} brings up several universities' intro-genetics webpages about yeast tetrad analysis! With clear, coherent explanations and well-drawn diagrams! UC Berkeley (pdf), Indiana U (pdf), U of Saskatchewan, and U of Rochester, just to name a few.
(Even better, some of those are straight-up webpages. Better than everything being PDF, although not better than everything being .doc, which sometimes happens.)
I often hear, second or third hand, how people from other schools use MIT OCW all the time. And I'm not denying that OCW is awesome. But it's not perfect, and there are other places to go if the OCW explanation of a topic happens to be consistently lousy.
The past few days I've been stressing over various topics from my genetics class, mostly yeast tetrad analysis. Going to lecture hardly helps; for this class, it's been generally true that the more confusing topics are very poorly explained. Usually the textbook does a good job of explaining things, but it doesn't cover yeast tetrads hardly at all. And I have a test in two days.
(Yeast tetrad analysis is pretty interesting, by the way. I might write a post about it if I ever actually understand it.)
Turning to OCW didn't help, because more or less the same professors have been teaching the class for N years, so the lecture notes are exactly the same. I can get practice problems (old homeworks and exams), which are very useful once I actually understand something, but not in this case.
So I turned to Google. And lo and behold, googling {yeast tetrad analysis} brings up several universities' intro-genetics webpages about yeast tetrad analysis! With clear, coherent explanations and well-drawn diagrams! UC Berkeley (pdf), Indiana U (pdf), U of Saskatchewan, and U of Rochester, just to name a few.
(Even better, some of those are straight-up webpages. Better than everything being PDF, although not better than everything being .doc, which sometimes happens.)
I often hear, second or third hand, how people from other schools use MIT OCW all the time. And I'm not denying that OCW is awesome. But it's not perfect, and there are other places to go if the OCW explanation of a topic happens to be consistently lousy.
Thursday, August 21, 2008
To be intellectually uncomfortable
From one of Uncertain Principles' link dumps comes this piece from Inside Higher Ed: Tolerant Faculty, Intolerant Students. Apparently, despite the myth of the politically stubborn professor forcing his views on poor openminded students, the truth is more like students haranguing each other and professors doing fairly well at not inappropriately expressing political views. (Well, at least in Georgia. The study might have turned out differently if done on CRAZY CALIFORNIAN COLLEGES!!!!1!11!one.)
(I should note that I have only been in actual political discourse once all year, and it was with a student on my hall who fervently supported Ron Paul. Perfectly friendly, civil discussion; I didn't enjoy it because I don't enjoy political discourse, period. I haven't seen any politics in classes, probably because I haven't taken any classes where it's even marginally relevant. Coulomb's Law? Politicize that, bitches.)
The thing that really struck me about this article, though, was one particular quote: universities are a place to go to feel uncomfortable intellectually. Obviously, the article means this with regard to one's political beliefs -- but it's applicable in a much larger sense.
When I encounter something I can't understand, something really confusing, I get anxious and upset. This happens a lot because I'm at a difficult school, and it's to be expected that I will encounter confusing things. But why should I get upset? This is an artifact of tying understanding to Success and Achievement (TM), rather than understanding for its own sake. Even though I went to one of those hippie elementary schools where there are no grades, I suppose I still have kind of a hang-up about 'succeeding' versus being way outside of my comfort zone.
I really admire people who respond to confusing things by first feeling humble, and then feeling happy that they have another puzzle to solve. People who get suspicious if it looks like they understand everything. It's not that I think such people never get frustrated, but that seems like a highly useful mindset for a scientist or intellectual, much more so than getting upset and frustrated. (Certainly, given the complexity of the real world, if you think you understand everything about X, you're almost certainly wrong.) This mindset should also make for a happier person overall. Apart from that, it feels like the right thing to do -- it appeals to the idealist in me.
Universities are a place to go to feel uncomfortable intellectually.
The world is a place to go to feel uncomfortable intellectually!
I'm going to try and embrace this mindset, embrace this mantra. A form of "intellectual asceticism, perhaps?" Hopefully I can change my knee-jerk reaction to the difficult and the confusing.
(I should note that I have only been in actual political discourse once all year, and it was with a student on my hall who fervently supported Ron Paul. Perfectly friendly, civil discussion; I didn't enjoy it because I don't enjoy political discourse, period. I haven't seen any politics in classes, probably because I haven't taken any classes where it's even marginally relevant. Coulomb's Law? Politicize that, bitches.)
The thing that really struck me about this article, though, was one particular quote: universities are a place to go to feel uncomfortable intellectually. Obviously, the article means this with regard to one's political beliefs -- but it's applicable in a much larger sense.
When I encounter something I can't understand, something really confusing, I get anxious and upset. This happens a lot because I'm at a difficult school, and it's to be expected that I will encounter confusing things. But why should I get upset? This is an artifact of tying understanding to Success and Achievement (TM), rather than understanding for its own sake. Even though I went to one of those hippie elementary schools where there are no grades, I suppose I still have kind of a hang-up about 'succeeding' versus being way outside of my comfort zone.
I really admire people who respond to confusing things by first feeling humble, and then feeling happy that they have another puzzle to solve. People who get suspicious if it looks like they understand everything. It's not that I think such people never get frustrated, but that seems like a highly useful mindset for a scientist or intellectual, much more so than getting upset and frustrated. (Certainly, given the complexity of the real world, if you think you understand everything about X, you're almost certainly wrong.) This mindset should also make for a happier person overall. Apart from that, it feels like the right thing to do -- it appeals to the idealist in me.
Universities are a place to go to feel uncomfortable intellectually.
The world is a place to go to feel uncomfortable intellectually!
I'm going to try and embrace this mindset, embrace this mantra. A form of "intellectual asceticism, perhaps?" Hopefully I can change my knee-jerk reaction to the difficult and the confusing.
Friday, August 15, 2008
New Look & Feel!
So I changed the blog template.
The main thing that bothered me about the old template was that the main text column, the most important part, was of fixed width. Which is annoying if you have a wide monitor. With this new template, go ahead and change the width of your browser window, and the Dendritic Arbor will change right along with you. We strive to provide a customizable reader experience. We strive to be adaptable, plastic, and dynamic, like actual dendritic arbors.
(It also bothered me that approximately 80,000,000,000 other people are using the old template. Then again, this is Blogger, what was I expecting, hmm?)
I chose this particular theme via the complex and deliberate heuristic of seeing which one looked the most like the one Larry Moran picked for Sandwalk. I've been reading his series on protein structure and admiring Sandwalk's look-and-feel. I guess this is the exact same theme as Sandwalk has, just with different colors.
By the way, the protein structure series is excellent and here are links to all its items. There isn't really a 'best' order to read them in, but I've put them in an order that sort of makes sense.
Evolution and Variation in Folded Proteins
Levels of Protein Structure
The Alpha Helix
Beta Strands and Beta Sheets
Loops and Turns
Examples of Protein Structure
The main thing that bothered me about the old template was that the main text column, the most important part, was of fixed width. Which is annoying if you have a wide monitor. With this new template, go ahead and change the width of your browser window, and the Dendritic Arbor will change right along with you. We strive to provide a customizable reader experience. We strive to be adaptable, plastic, and dynamic, like actual dendritic arbors.
(It also bothered me that approximately 80,000,000,000 other people are using the old template. Then again, this is Blogger, what was I expecting, hmm?)
I chose this particular theme via the complex and deliberate heuristic of seeing which one looked the most like the one Larry Moran picked for Sandwalk. I've been reading his series on protein structure and admiring Sandwalk's look-and-feel. I guess this is the exact same theme as Sandwalk has, just with different colors.
By the way, the protein structure series is excellent and here are links to all its items. There isn't really a 'best' order to read them in, but I've put them in an order that sort of makes sense.
Evolution and Variation in Folded Proteins
Levels of Protein Structure
The Alpha Helix
Beta Strands and Beta Sheets
Loops and Turns
Examples of Protein Structure
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