hobglobin

have you monitored the temperature of the gel during the run? you may be melting the pcr products while they are traveling through the gel.

you can try a denaturing (urea) gel to determine if your pcr product is specific.

LIES AND DAMN STATISTICS!!!!


WHen will biologists learn to do proper statistics?  I don't doubt at all that many published findings are not repeatable, and it's simply the fact that many biologists don't know how to properly do statistics.  I don't think it is out of anything nefarious (data manipulation etc.)  

Ask yourself, what does statistical significance mean?  If you can't answer that properly, go back and look back at a basic biostats book.  Statistical significance does NOT mean the following:

-that your results are reproducible (in fact statistically significant findings may only be reproducible 50% of the time)
-that your research hypothesis is indeed valid.  This one is a biggie.  Suppose a researcher has a hypothesis that A affects X which causes Y.  The researcher manipulates X and sees Y change.  Therefore the researcher concludes that theory A is supported since the null hypothesis is rejected due to "statistically significant data".  The researcher is in fact wrong, and falling into a fallacy noted by Aristotle 2000 years ago.  Statistically significant findings don't automatically mean A is true, since theories B, C, D, E, F.....could all say X affects Y as well, and may even be better at it than A.  A good scientist would therefore pit theory A against theories B, C, D, E, F....  but that in most cases is impossible.  Statisical significance ONLY describes the probability of observing your data under the assumption that your null hypothesis is true.
-that the probability that your hypothesis is true given your data D is answered by statistically significant data.  
-that your findings are even important, it has nothing to do with the magnitude of effect one observes.

Power of analysis should always be done to design an experiment to properly determine sample sizes needed.  Statisticians secretly laugh their asses off at much of the bio related literature from the poor handling of data and experimental design.  Underpowered experiments run the danger of not only type II statistical errors, but also type I.  It's laughable that hardly any biology studies outside of the clinic do not do power of analyses.  Journals need to start rejecting manuscripts without proper a priori experimental design.  

Here's a great article on the topic why most published findings are wrong:  
http://www.plosmedic...al.pmed.0020124

If you want to do much higher quality research than the 90% of scrubs out there properly design your experiments using POA.  Analyze your statistics for not only significance BUT ALSO MAGNITUDE OF EFFECT.  Use confidence intervals (SEMs should be banned from literature) and look at things like Cohen's d, eta^2, etc.  Maybe even try Bayesian techniques.

Another game for the Chit Chat section... Guess the quote !
Here are the rules:

1. The quotes to be guessed are from classic literature works, the work and the author should be internationally well-known.
2. The person who guesses correctly will post the next quote for guessing.
3. After 3 posts with wrong or guesses or cluelessness, the poster of the quote should give a hint.
4. Honor code - we won't type the quote into google and look for the answer this way. This is supposed to be a guessing game, and
    simply looking up the answer would spoil the fun, so please refrain from doing that.

Hope you'll join and have fun! So let's get it started with a hopefully not too difficult one :

"I do not know my age, for I have not counted the years I have been here."

As you probably have noticed since this morning, BioForum is loading much faster than before. This is because we have just migrated bioforum to a new dedicated server with much better hardware configurations.

Cheers,

bioforum

Yes, prot K degrades RNAses. It's better to add RNAse first and then prot K.

Read this: http://www.ncbi.nlm....v/pubmed/153663

You are not the first person to ask this question.
http://www.protocol-...osts/21549.html

But also keep in mind that digestion of DNA by DNAse (in contaminated samples, tips, microtube) is not as rapid as digestion of RNA with RNAses. Therefore, you don't have to worry about that and you have time to add your prot k and RNAse. Also, prot k might need time to degrade RNAse, so even if you add them together, RNAse A will digest your RNA before prot k digests it. You need to realize you are adding non-cellular RNAse A and prot k. They are excess in your sample.

You can also try this by yourself. Extract total RNA and prepare these samples:

1- total RNA
2 - total RNA + RNAse A, incubate (10-30 min) + prot K
3 - total RNA + prot k + RNAse

run on gel and check if you see any RNA smear or 18s/28s bands in the 3rd sample. If you do then prot k degrades your RNAse, although I doubt it will completely do that.

i know exactly how you feel! the only solution is a good preparation of your talk and most important, training, training, training, training. maybe you can start with a small audience, 2-3 people of your group, then increase the number. worked for me.

Good advice here but as a reminder - don't go mad.   EtBr is potentially dangerous and is a potential mutagen.   I'm not saying that you should be worried but take the precautions that you should (PPE, et cetera) and treat the stuff with respect.   A small dose is likely to cause no problems, but, as you'll know there isn't a predictable relationship between small doses of most mutagens/carcinogens and the long term health risk.
I shouldn't imagine that skin is particularly permeable to EtBr, so if you get some on you then get it washed off immediately with copious amounts of water and then carry on.
So, as long as you take the suitable precautions and behave sensibly you'll be fine.

Fun fact:  During the Victorian period people used to spike alcoholic drinks with chloroform.   I think that it exacerbated the effects of the alcohol.
And the cautionary tale...all those Victorians are dead now.

Dr. DoLittle in my lab

The usual suspects in my lab

(these are cracking me up, you guys , more than they should, I think I need sleep!!)

I am not kidding!
check here (not sure if this is already shared here)
http://www.sciencema...55/521.abstract

  You can say idealist or whatever, but you have to believe in what you do and that is worthy, otherwise how you can live in world where everything sounds fishy. There is always ups-and lows, nepotism, different countries different policies, many times we have to leave our country for sake of job (poor country or rich but not rich enough to accept u) or some injustice happens and we do not get particular job. Everybody perceives things differently, based on what surrounding person is.
But isn't that you get job/post-doc at particular place because you know something? Till now whatever you are, you are result of your work. now you started perceiving things in different terms but that should not undermine what you are.

Curtis, on 14 September 2012 - 06:22 PM, said:

nope, I don't come from a 'third world' country so you are right...but you're also not coming from a country where you don't know where your next meal is coming from or what clean drinking water is or where kids have to walk 2 hours to school and when it rains, there are even no roads to walk on... I have encountered misery, hopelessness and desperation (here and abroad so I have a fairly good idea) and I really wonder howcome a few have so much and so many have so little....life is unfair, yeah right....

But back to the foreign scientists diaspora, a big part of the cause is our gov'ts immigration policy of 'accepting only highly qualified and skilled workers' so it's easier to apply for permanent residencies or work visas..we say 'we have opportunities and you are welcome' ...we are an immigrant-based society and still is open for immigration and we're the poster country for multiculturalism....we favour diversity over assimilation and so most students and scientists who come here oftenly stay on working and living here and this is beneficial for us..it's our brain gain..but at the expense of other countries'.

I am not saying you shldn't come here or that you shldn't stay (if you're already here) bec if you can, then you shld if that's what you really want. But what if it's not possible and you have no other choice but to go back to your country (or stay there if you can't go out)? Then perhaps a change in attitude and goals is in order? I hear a lot of whining from some people I meet here- 'our gov't is corrupt, there is only chaos, people are lazy or inefficient, the streets are dirty etc etc' but who's gonna make it better...who shld care most about what happens to a country......not the international community (unless there's civil war or famine- but sometimes not even in these circumstances) ?

I'm sorry if I came off as being judgmental and for what you think as my harsh comments and questions..I was just trying to give you a different perspective esp now that you don't know what you're gonna do next with regards to your career and the high possibility of you not going abroad for a postdoc...it was actually a motivational piece to challenge you and encourage you to be Newton's gigantum blablabla ...(I tell this to myself whenever I think of 8 months of cold and ice) take it easy dude...btw..haven't I already told you that you're doing a great job as a mod? I did eh?

Nephrite, on 11 September 2012 - 06:17 AM, said:

Hi Nephrite,

I look at this differently...you shld be extremely proud of what you've accomplished all by yourself (of course with the help of your professor), despite all the challenges you encountered. And now that you've returned to your country, you should be able to find inspiration in the people that you care about, in the places that you love, in the things that you missed when you were out so this should actually motivate you more. You shldn't cop out otherwise, everything that you've endured would be for nothing. You've got a PhD you worked so hard to get. You lived in a foreign country and survived (how many in your country had this opportunity?)

You're feeling down and very tired now so you're allowed to take a well-deserved rest but once you have regrouped you'd realise that you will never allow yourself to become a useless member of society. Hopefully these feelings of depression and lack of motivation are only temporary but if they persist perhaps you shld also consider consulting a mental health professional.

If you really don't want to do research anymore, as the others have suggested, there are other career directions that you can take. But is the level of research in your home country comparable to the one you left behind? Perhaps there would only be simpler hypotheses to prove, cheaper techniques to employ etc. And are there many PhD degree holders as yourself? If not,then you definitely have an advantage so don't sell yourself short (and cleaning houses shldn't be an option cos you would probably suck at it ). Be strong and good luck.

Without wanting to get things started on the wrong foot, but I just can't help myself here , ascacioc, isn't your attitude a bit closed minded? ("...how can you be a scientist if you are not open minded? (I cannot see how somebody who believes in God is open-minded)."). Not saying anything, just saying...

I think both curtis and ascacioc are forgetting a very significant factor: the hardline creationists who hold to a literal 7-day creation are not the major voice in Christianity.I am convinced that they are misusing the text as an argument "for" just as much as hardline evolutionists misuse the text as an argument "against". Remember how I said that the text was written 3500 years ago, before the modern understandig of the world? Clearly, that means evolutionists cannot use the text as a proof text by comparing what was written with what we now know (For myself, I can't see how an old earth with the evolution of species can be wrong). It also means that creationists are mistaken when they try to make the document (which was written as Hebrew poetry)  into a modern historical summation of how things have come to be as they are.

Seriously, this is a total red herring when it comes to reasonable discussions about religion. No religion at all is centred on its creation narrative, but on the teachings of its leaders. As I said before, Christianity is not a philosophical position: anyone who tries to argue through it or against it (or for it) as though it is a philosophical position is going to come to false conclusions. The only way to get to know what Christianity (or Judaism, or Islam, Buddhism, Hinduism, or even Flying Spaghetti Monsterism!) is about is to actually do the research yourself and read the book, in your native language. As you say, ball's in your court...

ascacioc, it is a great ptiy that you were told such a simplistic statement about an atheist being someone who believes in evolution. Even more than that, your fellow PhD student gave a very poor response to you. I am most glad though that you were not involved in my PhD, nor that of people like Lennox and Collins... And I will lob the ball straight back : when God created life, he introduced differences in the 16S RNA as a means to separate the different species. (That last point was made tongue in cheek...)

I am not an expert on statistics but I think that the test depends on what do you want to know. I mean, correlations usually are used when you want to find associations between two (or more) variables (let´s say, phosphorus concentration and growth of algae, for example).

Do you want to know if there are differences between your treatments? If that is the case, you can use a t-student (if you want to compare only two groups, lets say treated vs  control) or better a one way anova.  But, as I said, it depends on what is your aim and on the set up of your experiment. If you can provide more information, maybe I can write a more specific example on what test to use and why!