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fibonacci

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Everything posted by fibonacci

  1. I'm trying to figure out how to sequence a gene I'm interested in from mRNA. Typically the way the lab does qrtPCR is they first make cDNA. using Applied Biosystems high capacity RNA to cDNA. Does this create full length cDNA of every RNA? For example, one gene I want to study is 5000 bp long in its mRNA. Does the reverse transcriptase make a full length cDNA? Next, what do I do with the cDNA? If I design primers using standard protocols that use online software, it gives me a pair of primers that are somewhere in the middle of the cDNA. Once I do PCR, will it amplify the whole cDNA or just the portion between the two primers? The problem is that I want the whole cDNA sequence to be amplified and sequenced. The entire sequence is already known for the mRNA, if I just designed primers that work all the way at the end of the 5' and 3' ends, would I get the entire sequence amplified using regular PCR? Would it have trouble amplifying a mRNA with 5000 bps? Would RACE PCR be appropriate here to create the entire cDNA amplification or is it not needed since the entire gene sequence is known for cDNA so I can get away with just regular PCR? Thanks in advance for any help. The reason I want to sequence the whole cDNA from mRNA even though the sequence for cDNA is already known is to look for mutations.
  2. Good points. I agree, magnitude of effect and biological importance should be the most important concept to analyze for a biologist, but too often journals won't accept a manuscript if they don't see "statistical significance" , which in the end is meaningless without effect size. If one knows what statistical test they want to run on the data that they are going to obtain, I was under the impression POA should be used to determine how many times to run the experiment. Why is it then so many journals accept n=3 without justification for 3? I don't understand the rule of thumb I guess if there's simply so much other literature out there describing the importance of enough statistical power in fields like psychology, clinical medicine, drug trials, etc.
  3. I don't think each cell counts as an individual "n". See: http://labstats.net/articles/cell_culture_n.html N should be the number of times your run your experiment independently. For example, let's day I do the tox test above on the cells day 1 with technical triplicates, repeat again on day 2 w/ technical replicates, and repeat again on day 3 with technical replicates. My n is still only 3 even if each sample I tested on those days contained millions of cells.. I've never seen a literature example where they run an ANOVA on a n=millions of cells. Cell culture cells all come from the same cell line and are all tested at the same time. Mice, however, are separate biological entities that are not the same, and can be tested independently. Testing all cells at the same time means they aren't independent because the same test was done on them all at the same time. That's on top of the fact they're all from the same culture. Again, in order to properly rule out error, you need to know how many times to run a test, which is "n". That's what power of analysis is for. So if that's what POA is for, then why is triplicate automatically assumed to be acceptable?
  4. Ok, for a very easy example: Let's say I want to test drug X on cancer cells to test for toxicity. I test 0, 50, and 100 uM of the drug on the cells and then count them to determine toxicity. The typical way to complete this experiment would be to repeat this experiment 2 more times and then run ANOVA or some other statistical test to determine statistical significance between concentration and cell count. This would be acceptable for many journals. What I'm hung up on, is why is n=3 by far and away accepted as a default number of times to run an experiment like this? Shouldn't one do a power of analysis to determine how much of a sample size you'd need to perform the experiment and have enough data points to run a proper ANOVA? However, if I were to test drug X in mice at different concentrations, then it would probably be absolutely required by an institutional board to conduct a power of analysis to determine how many mice I'd need so that I'm not unnecessarily killing too many mice or to determine if I'm not using enough mice which would result in my data being worthless. My question I suppose is, what makes cells different than the mice? Also, one other thing to mention--is I don't think 'data is just data' and that it should be up to the reader to determine if it is useful or not. The problem also with underpowered studies is that it can propagate type II errors, once a study is published with a type II error and it is repeated in literature, it can gain a foothold and be established as a scientific 'fact' when in reality, the results from all of the underpowered studies that replicated the original results are wrong because they mistakenly made a type II error due to lack of power.
  5. Right, experiments are done in triplicate so that statistical tests that generate P values can then be used to show "statistical significance". However, if your study is underpowered statistically, you run into all sorts of problems with type II errors and over estimating the mean effect size from an experiment. I'm under the impression that in cell culture work, each cell doesn't represent an independent measurement. Anytime you obtain a sample and assay your cells, the sample as a whole only counts as one measurement (technical replicates do not count of course). Am I confusing something here? I don't understand why many branches of science require a priori power of analysis while other fields can get away with simply doing triplicates.
  6. I don't understand why many biological experiments in literature are automatically run in triplicate. What's so special about the number 3? Why is it automatically assumed that a study run in triplicate has enough statistical power to make the observations from that study meaningful? Don't you have to run power of analysis first before determining proper sample size? Why is "triplicate" a mindless automatic default for number of times to do an experiment? I even see papers that get published in Nature and Science with experiments "done in triplicate". I don't understand why so much of the literature out there talks about the need to do power of analysis for proper sample size (in fields like clinical medicine etc.), yet many other branches of science can simply get away with the de facto default of "triplicate" and conclude that results from such an experiment are meaningful. Why doesn't all science require power of analysis to design an experiment?
  7. Let's say you messed up big time and took a course that is impossibly difficult to pass with the minimum grade. Is it bad to withdraw from a class in grad school? Let's say I don't even give a crap about teaching in academia after getting a PhD and that I can take a summer course to supplement the credits needed. What would you do if you knew you couldn't pass an insanely hard class that's not required but one of your electives?
  8. I've lost 22lbs while in grad school thus far, not because of some strict regimen of working out and dieting, but because of the fact that I simply don't have time to eat. Many days I only eat once per day. I wasn't that big to begin with, just slightly overweight (161 lbs for a male) and am now down to 139lbs. I haven't been this light since my freshman year in high school. At this rate, ill be sporting a 6 pack soon all without even trying, and ive never had a 6 pack in my life. Anyone else losing a ton if weight because they slend more time doing work than having time to eat?
  9. Know how you feel. I'm older too, and went back to school after a half decade in industry. Your academic skills definitely deteriorate while working BUT you have working experience which most lunatics in academia have never had in their life. No one really cares about your grades in grad school, just pass the courses you need to and do interesting research. If you're getting A+s in your classes, that means you are spending too much time doing class work and not enough on research. Sometimes I think the undergraduates in some of the classes I take are way smarter than me, but just I realize that they've never had a job in their life and have a hard time sifting out the useless academia trivia from what's really important to know. Grad school isn't about good grades like high school or undergrad college, it's all about the research.
  10. The postdoc we have in lab is starting to drive me crazy. She now thinks someone is sabotaging her experiments when she isn't around, and has now started whining to the PI about it. I know she's probably narrowed it down in her head to either me, or another guy in the lab. It just irks the sh!t out of me, that I'm probably being falsely accused of sabotaging her experiments behind my back. Now that she thinks sabotaging is going on, she's moving stuff around the lab and taking over cabinets and putting locks on them so someone can't "sabotage" her experiments. NO ONE IS SABOTAGING HER EXPERIMENTS. I don't understand where this bat sh!t craziness comes from. She's manufacturing completely unnecessary drama that's utterly absurd. I have never been around someone so toxic before at work. Not even in industry have I ever encountered someone so difficult to work with, so paranoid, and that's so against teamwork in order to take all credit for herself. Anyone else have a toxic lab coworker? What did you do?
  11. It's time universities should start creating their own open access journals and peer review them among themselves. The publishing cartels charge outrageous fees that do nothing more than increase tuition costs for everyone. The vast majority of the work that goes into academic journals mostly come from tax payer http://www.guardian.co.uk/commentisfree/2011/aug/29/academic-publishers-murdoch-socialist
  12. Look at that, Bank of America just dumped $75 trillion in derivatives liabilities onto the US tax payers: http://seekingalpha.com/article/301260-bank-of-america-dumps-75-trillion-in-derivatives-on-u-s-taxpayers-with-federal-approval This country is so screwed beyond belief because we let a few wall street banksters and politicians in Washington get away with rigging the system in order to makes millions, if not billions, on it for their own personal selves while it costs us a country as a whole trillions in losses. LIke the author said, these people privatize profits, but socialize all of the losses. These criminals should be tried as a racketeering ring under RICO laws. BoA dumping this kind of liability on the tax payers after a bailout that was only 4 years ago should be even more reason to protest for reinstatement of Glass Steagall.
  13. And it has only been 7 weeks in. I'm struggling with course work right now because I really don't see the point of all this BS. Course work is such a stupid formality, I just hate how I can't get to work in the lab right away because we all know that's what I'm there for anyway. Just learn the science you need as you go along with research, why all the useless coursework that you'll probably never or hardly use again? Also, sometimes I bother wondering why I went to grad school anyway. The economy sucks, and it will for a LONG time. We're training way too many PhDs for number of positions available in academia (which I plan on staying far away from). All manufacturing jobs and R and D are being shipped over seas. I'll be educated all right when I leave, but I'll still be poor. I don't see the point of blasting my brains out when it is increasingly likely that none of this BS will pay off. The whole system is f$@%ed beyond belief.
  14. if you haven't already: http://www.miller-mccune.com/science/the-real-science-gap-16191/ That article also recently won the American Association of University Professor's award for excellence in coverage in education.
  15. It's because the majority on here don't want to believe that the mantra they've been fed their whole life--that more education is always better no matter what the cost--is really not that true at all. Next year when the department of education starts tracking default rates on student loans out to 5 years the default rate on student loans is projected to almost double from 7% up to 14%. Only 40% of student loans are currently in repayment, the rest are either in deferment or are in default. The US has a massive ticking time bomb with regards to education and debt. The numbers don't lie, education costs keep soaring while jobs that pay livable wages continue to be off shored or are disappearing all together as the manufacturing base in this country declines. I bet the vast majority of kids on these boards have never even had a job or even looked for one. They have no idea what's waiting out here for them once they get out. Right now we live in a time when we are sending the most kids ever in the history of this country on towards higher education while the US economy is suffering from severe systemic and structural problems that will take decades to fix (a lot of problems may not be able to fixed at all). Suggesting that people should go to college or grad school no matter at what cost, because more education is always better, is completely stupid, especially when the economy is increasingly unable to absorb the huge swaths of new grads and pay them livable wages. College these days for many kids leads to nothing more than underemployment or temp jobs with no benefits with tons of student loan debt. Grad school just prolongs the underemployment while the interest builds on the principal and then you're caught in 20+ years worth of student loans. The whole f^cking system is one giant scam.
  16. The jobs that are being created by this economy are for the most part non exportable service and construction jobs, not jobs which require a college degree. During the years of 01 and 06, when times were still "good" and we hadn't entered into a Depression, the information sector of the US economy lost 645,000 jobs or 17.4% Computer systems design and related lost 116,000 or 8.7% of its work force. During that period of time, Oracle moved 2k jobs to India, among others including WS firms which moved their back room tech and analysis work overseas as well. And this was during the time when this economy was booming. Engineering jobs in general are also falling, due to that fact that manufacturing sectors that employ engineers are in decline. Again if you bothered to look at the period of time when things were "good" (01-06), the US lost 1.2 million jobs in the creation and building of machinery, computers, electronics, semiconductors, communication equipment, electrical equipment, motor vehicles and transportation equipment. The BLS payroll job numbers show a total of 70,000 jobs created in all fields of architecture and engineering, which also included clerical staff, for that period of time. So when we were booming and money and wealth was supposedly flowing, this economy created a mere 14,000 jobs per year in arch/engineering. The annual graduating classes for those majors is far in excess of that, and that before the FEDS allowed a minimum of 65,000 h-1B visas annually for skilled foreign workers as well as many more L-1 visas. All of the occupations with largest project employment growth in terms of the number of jobs for the next decade are in nontradable domestic services, meaning for this "incredible job machine" otherwise known as the US economy, are retail sales, registered nurses, postsecondary teachers, customer service representatives, janitors and cleaners, waiters and waitresses, food preparation, home health aides, nursing aides, orderlies and attendants, general and operations managers. Few of these jobs require a college education. People are propagating a myth that education is an axiomatic good, one that many students believe and that is leading to economic ruin. As a percentage of population, there are more people in college than at any time in the history of the republic, and precisely at a time when the economy is incapable of creating high wage, high skill careers which cannot be shipped off overseas, or are not being filled by foreign workers. To insist that college is somehow going to create the income when those jobs cannot begin to pay back high debt is plain ridiculous....
  17. Yeah, run for cover. These days you are lucky if you can get a $50k a year job with a PhD. The US economy is absolutely hemorrhaging high tech jobs and other well paying work like a slit wrist. Once we get out of this depression most of the jobs that will have been created will be in retail sales, janitor and cleaning services, food prep, RNs, nursing aides, and other low paying service jobs that can't be outsourced. Many new jobs won't need even a college degree. It's highly likely you are probably better off with your masters than a PhD.
  18. I don't understand what students mean when they say they want to work in "industry" after they get out when they're asked "so what are your future goals and career aspirations?" Basically it means they have no idea what the hell they're doing and they're just hoping, maybe even expecting, that there will be a job there for them waiting when they get out. Sorry, a PhD after your name doesn't mean there will be anything there after you graduate. Being honest with yourselves, how many of you are hiding out there in grad school really only because you have no idea what you want to do with the rest of your life, or because you didn't want to get a job, or because you couldn't find a job? I laugh every time I hear someone say they want to work in "industry" when they get out, that's like saying "i like food".
  19. Been trying cold drip brewing recently. Much much smoother, less bitter, and less acidic. You can definitely tell a major difference. Some people won't like a cold brew while others may vastly prefer it.
  20. fibonacci

    Bikes

    ^ nice bike. Fixie skid stop
  21. fibonacci

    Bikes

    I don't see why this wouldn't be a decent fixie bike for the average commuter (they even have one for short people): http://www.amazon.co...07679311&sr=1-2 They have a red one with straight bars if you don't want to get all low and in a race position with the drop bars. For $300, you probably won't be able to find a 'good' bike per say anywhere. You'll have to get an old one off of ebay or craigslist if you want better quality. Spend $250 and then take it to the bike store to get it converted to a fixed gear bike. Just be aware with a fixed gear bike, the pedals constantly move, you can't stop pedaling. It takes some getting used to, you won't be able to coast before trying to stop. Like others are saying, however, it has the advantage that you'll be able to stop in bad weather by simply just resisting the way the pedals are moving with your feet. Brake pads can fail if your rims are wet and it is less maintenance. Edit: here's one for $199 too and they have a 50 cm version: http://www.amazon.co...ref=pd_sbs_sg_5
  22. Just go into Calc 3. Calc III doesn't really emphasize tricky integrals it's more about the concepts behind using multivariables. Unless you have a huge jerk for a professor, you really won't be doing ludicrously difficult integration.
  23. fibonacci

    Bikes

    I'd highly recommend for most of you to fall in line w/ the hipster crowd and simply go with a 'fixie' road bike. I've fixed bikes for years when I was younger. The more moving parts a bike has, the bigger pain in the ace it is to fix it. Fixing something simple like a chain popping can be a huge pain on a bike that has multiple speeds. A fixed cog will be fine for probably 90% of people simply using a bike to just commute and get around town.
  24. It also doesn't help when you have multi BILLION dollar entities like JP Morgan, Wells Fargo, etc. rigging the system so that students can't get out of loan debt while simultaneously selling their debt on Wall Street to investors which is putting the pedal to the floor on tuition inflation. There are clear conflicts of interest at almost every single level with regards to student loans, lenders, and borrowers. I suspect that universities too may also be investing in SLABS, which is down right sickening. That would give them incentive to have their students in debt and to constantly raise tuitions. Amazing how the whole country has gone to hell since banks were deregulated in the 90s--.com bubble, housing bubble, oil speculators driving up prices, and a soon to be bursting student loan bubble.
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