Thursday, December 31, 2009
For those of you who care
...I'll note again, as I did at the inception of this blog about 3 years ago, that I also maintain a personal blog over at Livejournal. Also, there is an LJ syndicated feed of this blog.
Exposing oneself to disturbing things
[If anyone's here from Iron Blogger, welcome!]
Home for the holidays, I've been rewatching a few Firefly episodes with my family. Because everyone gets to pick, said rewatching has not consisted solely of "Heart of Gold" over and over and over :) -- and I end up watching the episodes with disturbing bits that I don't like.
What don't I like? I don't like stabbings or incisions (and I'm not fond of injections either, or at least the needle-entry part, though IVs are OK). I don't like torture scenes. I don't like when characters walk alone through dark creepy hallways where it's implied that Reavers or similar will suddenly jump out at people. I don't like half-rotted or mummified corpses.
I'm not sure what the common thread is between these things -- or between the things I don't mind (blunt trauma, gunshots... or most anything not listed above).
Somehow I keep making myself sit through movies that have this stuff. I managed to freak myself out pretty badly at a free showing of 28 Weeks Later a couple of years ago, when I could have just got up and left. Perhaps I feel it's unseemly for a grown, voting adult to have to not watch things. After all, when I was a kid I didn't have any compunction about leaving the room when it got to the Emperor-electrocutes-Luke scene in Star Wars. My dad's philosophy on scary things is that he leaves the room because "I don't need that stuff in my head, it's not worth it", which strikes me as very sane. What, after all, is the higher purpose served by my watching Mal and Wash get electrocuted repeatedly?
However, I feel that there should be a corresponding policy to not avoid watching (or reading about) the horrific things that go on in the real world, because in that case a horrified reaction can translate into a call to action (charitable giving, political action, whatever). This is actually one of the strongest arguments I've heard in favor of free speech, and in particular freedom of hate speech: let's get it all out there so everyone's at least aware that this sort of badness exists, rather than letting it stew in private gatherings until it becomes hate crimes.
Home for the holidays, I've been rewatching a few Firefly episodes with my family. Because everyone gets to pick, said rewatching has not consisted solely of "Heart of Gold" over and over and over :) -- and I end up watching the episodes with disturbing bits that I don't like.
What don't I like? I don't like stabbings or incisions (and I'm not fond of injections either, or at least the needle-entry part, though IVs are OK). I don't like torture scenes. I don't like when characters walk alone through dark creepy hallways where it's implied that Reavers or similar will suddenly jump out at people. I don't like half-rotted or mummified corpses.
I'm not sure what the common thread is between these things -- or between the things I don't mind (blunt trauma, gunshots... or most anything not listed above).
Somehow I keep making myself sit through movies that have this stuff. I managed to freak myself out pretty badly at a free showing of 28 Weeks Later a couple of years ago, when I could have just got up and left. Perhaps I feel it's unseemly for a grown, voting adult to have to not watch things. After all, when I was a kid I didn't have any compunction about leaving the room when it got to the Emperor-electrocutes-Luke scene in Star Wars. My dad's philosophy on scary things is that he leaves the room because "I don't need that stuff in my head, it's not worth it", which strikes me as very sane. What, after all, is the higher purpose served by my watching Mal and Wash get electrocuted repeatedly?
However, I feel that there should be a corresponding policy to not avoid watching (or reading about) the horrific things that go on in the real world, because in that case a horrified reaction can translate into a call to action (charitable giving, political action, whatever). This is actually one of the strongest arguments I've heard in favor of free speech, and in particular freedom of hate speech: let's get it all out there so everyone's at least aware that this sort of badness exists, rather than letting it stew in private gatherings until it becomes hate crimes.
Friday, August 7, 2009
Review papers are your friend!
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
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
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.
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