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fibroblast cells and fibers
I am interested in fibroblast cells in human arteries. Here are the things that I am not clear at the moment and I could not find any answer from the literature:

What are the dimensions of these fibroblast cells? How big are they in terms of diameter and thickness? How big are they if they are comparing with the collagen fibers in the artery?
I think they have to attach themselves to the fibers in order to proliferate or differentiate. However, I do not know, do one fibroblast cell attach itself to one fiber and then "crawl" to another? Or do they "sit" on a network of fibers?

I will appreciate your help on this and will be even more grateful if you could provide me with some literature that I could find the answer to these questions.

Regarding dimensions:

As per this image the length and breadth seems to be ~30-50 μm (area should be roughly around 900 μm²). The third dimension (thickness) as per this article can be assumed to be around 3-7 μm.
Regarding cell attachment:
Cells attach to extracellular matrix via integrins, which attach to ECM proteins like collagen and fibronectin. The matrix is a net of fibres and the cell is not specifically attached to a single fibre but rather "sits"(as you said) on the matrix [see the below image]. A single cell also simultaneously interacts with two different types of ECM proteins such as fibronectin and collagen.

During migration they move along the matrix using lammelipodia (actin filament polymerization in the direction of motion).
Fibroblast express ICAM1/VCAM1 (Inter/Vascular Cell Adhesion Molecules) under certain conditions such as inflammation and bind to T-cells and endothelial cells.

somehow the second image is not showing in the answer.. this is the link

I dont know exact references for these but you can find these info in cell bio books such as Molecular Biology of the Cell by Bruce Alberts

Hi WYSIWYG, thanks very much for your explicit answer! This really helps! Do you mind if I ask you another question: what are the large thick fibers and small very thin fibrils in the second image? Are they collagen matrix?

Also, note that the width and height of the second image is about 0.5 μm. So if a normal fibroblast cell is 40 μm in length, then this means a fibroblast should be 80*80 times of the size of the fiber network showing in the second image? Then How does it attach itself to such a complex network of fibers? Merely via integrins?

mostly integrins as I understand. But integrins are diverse.. some bind to collagen some bind to fibronectin some bind to laminin etc.


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Can parts of a human brain be asleep independently of each other, or vary in the times required for them to fall asleep?
I know that some birds and marine animals can continue complicated activity (swimming, flying?) while one hemisphere of their brain is asleep.
I'm interested if human brain has some parts of it that can be asleep while others are awake? In other words, can a human brain be only partially asleep while experiencing insomnia or similar sleep disturbances?
If human brain can have different parts "sleeping" independently of each other, is it possible that the times to "fall asleep" vary between these different parts of the brain?
I would appreciate research articles on the topic or just the names of brain regions that may exhibit behavior described above.
Update: I've taken a look at R&K "principles and practice of sleep medicine"' and it mentions the following parts as involved in sleep:
Medulla, preoptic area, hypothalamus, thalamus, entire neocortex involved in NREM.
Neurotransmitter systems: histaminergic, orexinergic, serotonergic, noradrinergic
Sleep factors: adenosine, interleukin-1 and other cytokines, prostaglandin D2, growth hormone releasing hormone, nitric oxide, all promote sleep in or around preoptic area.
This makes me hypothesize that drugs that modify the effects of these systems (ex- caffeine affecting adenosine) could result in sleep- related disturbances in these systems, potentially causing them to fall asleep later that usual. But I'm looking for more info to fihre out if this is true

Related CogSci question: http://cogsci.stackexchange.com/questions/3602/what-is-the-mechanism-behind-unihemispheric-sleep-in-animals

Reading K&R "Principles and practice of sleep medicine" 4th edition, on page 15, under sleep onset I've noticed the following paragraph:

Is "falling asleep" a unitary event? Our observations suggest that it
  is not. Different functions, such as sensory awareness, memory,
  self-consciousness, continuity of logical thought, latency of response
  to a stimulus and alterations in the pattern of brain potentials all
  go in parallel in a general way, but there are exceptions to every
  rule.

The book continues to mention that it is possible to drift in and out of sleep for a few seconds.
I've became aware of experiments on split brain patients (severed corpus colossum) which revealed that two brain hemispheres have their own conscious awareness. Some experiments even paralyzed one hemisphere to see how the other one responds. As such, it appears that a human can function with only one hemisphere active at a time.

I have often been aware that I am falling asleep, such as when I notice that my train of thought is no longer logical. Once, while meditating, I was still self-aware even though my body and the rest of my mind were asleep. Interesting.

Have you ever tried to read a complicated book after several hours of hard work that required a high concentration? Imagine that you have forced a particular area of your brain. After a hard work, the areas of the brain that we used to finish a work seem don't respond to any stimulation and make us feeling tired and as we can't understand what we are doing anymore. This happens because we have forced that particular area of the brain and it needs to reinforce the connections it made and to do this, it has to sleep, such as we do during the conscious (you know you are going to sleep) sleeping. Eventually we can say that some particular areas of our brain must sleep if forced to a hard work and they do it when you feel like you can't get something which you know you can. A recent study has demonstrated this by understanding physiological functions of sleeping.
Nature article of Vyazovsky V. et al. Vol. 472, pages 443-447, April 2011 (I am not copying the link anyway, have to search it by yourself). Or to Science of Bushey D. et al. Vol. 332, pages 1576-1581, June 2011. And on Nature Review Neuroscience of Diekelmann S. And Born J. Vol 11 number 2, pages 114-126, February 2010.

Can you add some evidence to your answer, backing this assertion up, such as the "recent study" you refer to?

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Is there any other function of DNA?
We all know that DNA acts as a genetic molecule. Does DNA have any other function in the cell other than being a genetic material and carrier of information?

In eukaryotes DNA has a structural as well coding function. Parts of chromosomes called centromeres bind to proteins and form a scaffold which helps chromosomes attach to each other and correctly segregate during division. Technically this is still related to the transmission of genetic information though.
Since RNA can act as an enzyme (the so-called ribozymes), scientists have hypothesized that DNA can also have an enzymatic function, but no such function has been found in nature. Artificial DNA enzymes (deoxiribozymes) have been designed to cleave DNA, covalently modify proteins, ligate RNA molecules, and even catalyze reactions between small molecules. See this lab's website.

as far as i know DNAzymes are purely synthetic.. Are there natural DNAzymes too ??

No, there are only synthetic ones

I want any other function inside living cells. Is there any ?

From what I researched, no

DNA has the following functions other than genetic material :

It can function as a molecule that attaches to TLR ( Toll like Receptor ) on or in cells and so  can contribute to innate immune response.
It can act as carbon source. ( We have nucleases enzymes in our intestines.)
It can be structural component of biofilms.
It can also influence pH in cells as it is an acid.
It is a structural component of neutrophil extracellular traps to wrap up microbes.

Please feel free to correct any errors I have made.

DNA has been shown to be important for biofilm formation in certain bacteria.  This is extracellular DNA that comes from cell lysis.

Interesting! Can you give a reference?

Heres the first one I could find that is open source, but if you search "extracellular DNA biofilm" in google theres loads of papers on it: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016861

Yes, maintenance of the genome.  But not DNA specifically, but DNA and all of its associated proteins.

Could you give a bit more detail here? It's hard to tell what you're referring to.

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What is dental health like in animals?
Do animals suffer from tooth decay, such as cavities? Is it to the same extent as in humans, or do animal teeth decay more or less? If teeth decay in animals as well, are there natural strategies animals use to heal it? And, lastly, how do animals take care of their teeth?

If this is in comparison to humans, the animals are more or less free from the two common dental diseases by which teeth are lost in humans. These are dental caries (dental decay)  and periodontal disease (gum disease).
Common to both the diseases is the biofilm which forms on the teeth known as dental plaque.
If the plaque is colonised by acidogenic (acid producing) micro organisms cavities are produced in the presence of refined carbohydrates.
The gum disease is produced by bacterial toxins from the plaque causing inflammation of the surrounding gum resulting in loss of supporting structures of the tooth including bone resulting in loosening and loss of teeth.
In animals in the wild, plaque accumulation is effectively controlled by the coarse unprocessed diet which has a very good scrubbing effect. Besides wild animals are not exposed to refined carbohydrates like sugars.
Hence wild animals do not have these two diseases. However domesticated animals on human diet are prone to similar diseases.
In wild animals severe attrition (wear) and fracture of teeth are more common.
Wild animals will not survive long without adequate number of functional teeth.

Human tooth decay is mostly from eating foods high in sugar.  So animals don't generally get cavities.  (Except for humans' pets.)  But animals can have other tooth problems.  For example, elephants grind down their molars from eating so much plant material.  Fortunately for them, they have a supply of new molars that come in regularly.  Unfortunately for them, that supply is limited.  When an elephant runs out of molars, it dies of starvation.  Another example is plaque on dogs' teeth, which can lead to gum disease just like in humans.

Outside mammals animals just let bad teeth fall out and replace them as part of a continuous cycle of losing and replacing teeth, they don't keep teeth long enough for it to be an issue. Mammals are... weird, they only get the two sets. I would go further but Ram's answer covers how this question applies to mammals quite well. Although I should mention humans have relatively thin enamel out diets are rather low demanding in no small part due to the invention of fire.

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Why Is The Toughness Of Skin Different On Different Parts Of The Body?
My cat was licking my arm with his sandpaper like tongue. It hurt and the area he was licking was slightly smarting afterwards. However, when he licks the palm of my hand the feeling is rather ticklish and results in no pain during or after.
We can pick up sharp, prickly, abrasive objects with our hands (palms/fingers) and even rub against them and have little to no damage incurred. However, if we do the same with say the back of our hand we are more likely to be injured.
I thought maybe the skin is thicker in our palms and that is resulting in our palms being more resilient. But, our palms are so sensitive to touch that it makes me think the skin is thinner to allow the nerves closer access to our environment.

Why is there such a stark difference in different areas of skin on our body when it comes to feeling pain and being susceptible to injury from scrapes, bruises, cuts, etc?

Sensitivity to touch depends on the density of mechanoreceptors.

@WYSIWYG do you want to post that as a more detailed answer so I can accept and close this?

As dd3 said the density of mechanoreceptors dictates skin sensitivity to touch (also look at penfield map. It is a nice illustration of how different senses are mapped to the brain and to what extent each region is sensitive to stimulus). Skin thickness is also different in different regions. This article says that Dikkopf1 (a Wnt pathway antagonist) controls skin thickness.

There are different receptors and different forces at play here.
Skin thickness depends primarily on frictional forces and is determined by the thickness of the epidermis, which is made up of 5 layers of keratinocytes:

Stratum basale
Stratum spinosum
Stratum granulosum
Stratum lucidum
Stratum corneum

The areas that are subjected to high frictional forces have the thickest epidermal layers, such as palmar surfaces of the hands vs the dorsal aspect of the hand (the back of the hand)which is significantly thinner.
The actual thickness of the skin doesn't always correlate with how tactile or painful stimuli are perceived. The receptors can be very sensitive even where stratum corneum is the thickest and vice versa.

Your links are for specific epidermal layers vs providing adequate citations for the claims you make throughout. Please edit.


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Why not use Cu(OH)2 as a dye for protein electrophoresis?
As I know, $Cu(OH)_2$ $+$ Protein $\rightarrow$ Purple. So why not use $Cu(OH)_2$ as dye in protein electrophoresis?

May be the OH-group changes the summational charge of the protein (which is very important for the separation), as by adding the compound, we change pH.
Still, copper is used in electrophoresis, but as CuCl2. http://www.ncbi.nlm.nih.gov/pubmed/2449094

Copper hydroxide does not dissolve very well in water, which means you will have trouble soaking them into your gels.
Also, even if you managed to add enough copper hydroxide beforehand (in the wells), it is unlikely that the copper(II) ions, if any existes, will travel along with the protein due to its size. (Copper hydroxide itself does not have any charge.)
As for the paper Stella mentioned, the copper salts are used after electrophoresis to produce a negative image, which is exactly what you cannot do with copper hydroxide.

Protein electrophoresis is often done in an alkaline pH (8.6), which is not exactly nice if you want to use $Cu(OH)_2$.
If you need quantitation, you can use Coumassie blue or Amido black. If you want sensitivity you can use silver. These existing stuff are quite OK.


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How do you remove DNA/RNA from plastics?
I want to know how to make plastic containers free of DNA & RNA? this is just for knowledge

Use disposable containers like Eppendorf tubes and Falcons, that is by far the easiest way.

Lab cleaning solutions can be bought which remove/destroy nucleic acids such as this one. UV is another technique for destroying DNA. And bleach is used to remove contaminant DNA in ancient DNA studies.
Searching for a DNA/RNA lab cleaning protocol would be a good idea to - something like this.

Both the 'this one' link is not working and the 'something like this' aren't working, would you mind providing updated links?


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What is the exact principle of capillary isotachophoresis?
I know it's a type of capillary electrophoresis, but I don't get how does the separation happen exactly.

Many of us are using isotachophoresis all the time without even realising it. The explanation that follows may not be exact, but I hope that it helps.
Firstly, think about a simple electrophoresis set-up - in agarose electrophoresis of DNA we have a uniformly distributed buffer such as Tris-borate. The current is carried by the borate ions and the DNA molecules run through the homogeneous electric field towards the anode. They separate by size because of the sieving effect of the agarose matrix.
Now let's think about what is happening in an SDS-PAGE separation (a Laemmli gel). Just to remind you:
The running (or reservoir)  buffer is Tris-glycine pH 8.3
The stacking gel is in Tris.HCl pH 6.8
The separating gel is in Tris.HCl pH 8.8
When the power supply is turned on, the glycinate ion in the top reservoir begins to move into the stacking gel. When it hits the zone of pH = 6.8 it is much closer to its pKa and so is now only weakly charged. Consequently it slows down. Meanwhile the chloride ions in the stacking gel have begun to move rapidly down through the stacking gel (towards the anode). This creates a discontinuous electric field: the chloride zone has a lower resistance than the glycinate zone so the field strength in the chloride zone is weaker than the field strength in the glycinate zone. This is a stable situation because the ions cannot stray into each other's zones: if a glycinate ion happens to diffuse forwards into the chloride zone it will experience a weaker field and slow down, whereas if a chloride ion diffuses back into the glycinate zone it will hit the stronger field and quickly move ahead again.
What about the SDS-protein complexes that were in the well? (I'll just refer to these as proteins from now on.) Their ionic properties lie between the glycinate and the chloride, and so they eventually end up moving through the stacking gel between the trailing zone of glycinate and the leading zone of chloride. Any protein that finds itself in the glycinate zone will move quickly forward (due to the higher field strength) and any protein that moves ahead by chance into the chloride zone will slow down due to the weak field strength, falling back into the protein zone. And in this way these zones move in a procession through the stacking gel, with the proteins being focussed into a very narrow region in the middle. The glycinate, the proteins and the chloride are all moving at the same speed, and so this is an example of isotachophoresis (isotacho- = "same speed"). The polyacrylamide gel matrix in the stacking gel has no separating function - its role is simply to stabilise against convective effects. The proteins can reach very high local concentrations in the stacking gel, and if you look carefully you will often see refractive effects as your sample runs through the stacking gel. In this way the protein sample is "applied" to the separating gel in a very tight band, perfect for achieving high resolution separation (bands broaden by diffusion in the separating gel so the tighter the bands ar eat the point of loading, the better the final result.)
Once the sample hits the separating gel, all of this breaks down: the pH is now 8.8 so the glycinate becomes strongly charged and zooms off ahead with the chloride through the gel, leaving the proteins to move more slowly due to the sieving effect of the polyacrylamide matrix.
It is possible to set up an isotachophoresis as described above in isolation. Individual proteins will have slightly different ionic properties and will line up in the region between the two buffer ions, allowing for their separation.


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Aside from cockroaches and other insects, which species (of all kinds) are 'immune' to ionizing radiation?
I have just been reading this excellent question and answer about cockroaches ability to withstand ionizing radiation.  My question is an extension of that question: aside from insects, which other animals (if any) have an increased defence against ionizing radiation?
Are there species of plants, fungi, and bacteria also "immune"?

Deinococcus radiodurans (bacteria), tardigrades

According to Paul Stamets, Gomphidius glutinosus is especially well suited to collecting Cesium-137:

G. glutinosus has been reported to absorb – via the mycelium – and
  concentrate radioactive Cesium 137 more than 10,000-fold over ambient
  background levels.

That article and Stamets' book Mycelium Running have more details on other species.


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What do you call the non-Wild Type allele?
Can I have a list of suggestions, such as the mutated allele, other allele, etc. ? Are there any blatantly missing ones on my short list?

List-type questions don't work well here. Your question is a bit borderline, so we'll see what everyone else thinks. One that you are missing is mutant allele.

If not wt then it is a mutant.

Usually its major allele and minor allele.  When you hear this language, you feel that the major allele is the 'wild' type, but its more precise to avoid 'wild'.
This language avoids the question of which one is 'wild' since different populations may have different ratios of the two.

This is a nomenclature I have almost only heard in bioinformatics context. In most biological literature I have generally seen "wild type" vs "mutant". Although the point about the different population is true one may argue that using major and minor brings confusion as the same allele can be major in a population and minor in another...

how do they know which allele is wild type?  you would only know if you were doing an experiment where you created the mutant yourself I would think.

I am not saying that wt/mutant is a better nomenclature in absolute, just that it is - probably for historical reasons - the one most commonly used (the wild type nomenclature if you wish :P). In general you would use wt for the allele most abundant in the general population.

Wild type refers to the phenotype of the typical form. Normally it is wild type is being compared to a mutant allele because the allele is not longer typical. So the answer would an allele that is not typical which, in most cases is a mutated allele.


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How to ligate DNA to an RNA molecule
Is it possible to ligate a small DNA molecule to mRNA?  I was reading a protocol from Current Protocols in Molecular Biology and one of the procedures involve ligating a DNA molecule to mRNA using a DNA splint that overlaps between the 5' of the DNA molecule and the 3' of the mRNA.
Would a reaction like the following work?
5'-phosphorylated DNA linker
mRNA library
DNA splint
T4 DNA ligase + buffer

T4 ssRNA ligase will work for this.. Check this page on NEB site.


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What happens to the holes in the skull where teeth were developing?
In Skeptics.SE, a question regarding the skulls of children arose. Depicting a scary skull where the permenent teeth are "hidden" inside the jaws and the milk teeth are in the their place.
After all milk teeth fall out, and the permanent teeth "rise", do the holes left by them filled up, or do we go around with holes in our jaws?
The question on Skeptics
The picture:

Permanent teeth have much larger and stronger cemented roots.

Jaw development is very prominent during the growth of a child. There are no holes that remain in the jaw after the emergence of all the permanent teeth. Permanent teeth are not fully formed and hidden. They develop while milk teeth, meanwhile do the temporary job.

When the tooth falls out you can see a little adult teeth behind it after a couple of weeks the adult teeth will grow and move to the front and the adult teeth will not fall out so look after your teeth for your life

Bone growth continues in the human body for quite awhile after permanent teeth come in. Generally, the complete closure of all growth plates doesn't occur until the mid 20s.
As the top and bottom primary teeth come in, the empty space left behind is gradually filled by growth of the maxilla and mandible, respectively. During the time period when teeth are actively coming in, teeth can displace into the empty space left by other permanent teeth that are no longer there, resulting in crooked teeth.

This is a skull of a child of around 5 plus years; based on the eruption status and is an example of  classic development and eruption pattern of of the teeth at that age. everyone goes through it. It probably looks scary to persons unfamiliar with normal anatomy and because the outer cortical plate has been removed to show the developing tooth germs. This is out of a anatomy museum meant for medical or dental students.
At no point of time during the eruption is a cavity left behind. As the tooth erupts the base of the crypt (cavity) gets filled up.


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Coiling of chromatids during cell division
What is exactly coiling of chromosomes?
I just heard about the names i.e paranemic, plectonemic, orthostichious, anorthospiral.
I have ecaxtly no idea of what phenomenon is this.
Also what type of coiling occurs during meiosis and mytosis type of cell division.
Could it be explained in detail....?


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How potent is the venom from the Australian platypus?
The Australian platypus, is a marsupial with a unique feature - in that the males of the species have venom sacs around their hind-claws.
My understanding is that this venom is not lethal to humans, however, how potent is this venom to other animals?
Also, how does the platypus 'manufacture' this venom?

The Internet says that platypus venom can kill small animals, but I haven't come up with a definitive source for this.
There is an informative Wikipedia page about the venom here. Basically the venom contains a number of toxic peptides including a family of C-type natriuretic peptides, some defensin-like peptides and nerve growth factor. Defensins are very widely-distributed in nature but typically are used by cells as antimicrobials. Natriuretic peptides have a range of effects upon fluid balance and blood pressure control - these are the best-characterized components of platypus venom.
These peptides are all genetically-encoded, so they are made as segments of precursor polypeptides. Intriguingly some of the the C-type natriuretic peptides contain a D-Leu residue. (All of the amino acids used in protein synthesis are the L stereoisomeric forms.) The presence of a D-amino acid in a peptide derived from a genetically-encoded molecule suggest that there must be a specific isomerase present, and evidence for this isomerase in the venom has indeed been found as reported in Torres et al. (2006) Mammalian L-to-D-amino-acid-residue isomerase from platypus venom. FEBS Lett.580:1587–1591. The introduction to this article states:

Platypus venom contains many non-protein and protein components...whose roles are yet to be established.


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Pump to separate protein/DNA instead of electrophoresis?
I know that DNA molecules or proteins can be separated in electrophoresis because the electrical charge is used to pull the DNA through the gel. So instead of using electrical, can I using physical impact like pumping ?

Do you have a particular reason for wanting to use pressure instead of electricity? Any specific application in mind?

Well ... A lot of reason why i think pressure is better than electricity. Pressure will work with any molecule even they don't have charged, so it give better result without denaturing the protein. It's also safe budget, safe time when protein electrophoresis need to using chemical substrate for denaturing the protein. Pressure also safety for a diy bio like me when i don't want to working with high electricity... And i think with pressure i have more and more application, may be a machine that can purify protein, detect viruses in real time from good protein separate result...

Much of this comment should be included in your question. It gives background and makes the question easier to answer, and also shows what particular problems you have been thinking and that you are looking for DIY solutions.

If you tried to set up a system in which movement of DNA molecules through a gel matrix was driven by pumping buffer, the gel would become compressed. I am old enough to remember running vertical agarose gel electrophoresis overnight, and these gels would collapse under normal pressure to about 2/3rds of their initial height. A gel porous enough to give useful separation of DNA molecules just isn't strong enough to withstand pumping.

not from gels but physical force can be used to separate molecules.. e.g. density centrifugation, filtration. Closest to what you are asking is size exclusion chromatography in HPLC where molecules are separated based on size and high pressure is maintained in order to pump the solution (containing the analytes) through the stationary phase.
As Alan Boyd mentioned, the gels cannot withstand high pressure. In HPLC silica beads are used (sometimes coated with specific resins for e.g. in ion-exchange chromatography).
The principal differences between electrophoresis and HPLC (chromatogrphy) are:

HPLC relies on differential interaction of the molecules with the stationary phase (i.e beads) whereas electrophoresis is purely based on the hydrodynamic size of the molecules.
HPLC uses pressure to keep the entire mobile phase (solution) pumping through the stationary phase, whereas electrophoresis uses electric field to move the molecules.
HPLC can also be used for uncharged molecules

Size exclusion and gel-filtration chromatographies resolve the molecules based on size as in the case of electrophoresis but as I previously mentioned, the molecules need not be charged. The techniques used for analysis of molecules in both techniques can be similar. For e.g UV absorption/fluorescence. An advantage of HPLC is that it can be connected to other analyzers like Mass Spectrometer, Flame Photometer etc.

Hello, thank you so much. What is the gel HPLC using for separate molecules ? What different between HPLC and Electrophoresis (i mean in the result)

edited the answer..

Hello, so if i replace the gel using in electrophoresis with the gel-filtration using in HPLC. That mean i can use pressure to separate molecules instead of electrical ?

yes. you can. but HPLC is a different setup altogether. there are pre-packed columns available, which contain the beads.

Amazing ! As what i think in my dream, there are a method like that. Thank you so much !

No, you can't use mechanical pumping for gel electrophoresis. The nature of electrophoresis is the create a uniform electric field through the gel, and this electric field stimulates motion from the charged DNA/protein.


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Why are enzyme-catalysed reactions slower at lower substrate concentration?
Suppose I'm using 200 nmoles of enzyme and 2 mmoles of substrate. The enzyme should be saturated but if I use 50 mmoles of substrate, the reaction will be faster. Why?
I just can't get it! Even at lower concentration of substrate the enzyme is saturated (all enzyme molecules are engaged in the reaction) but still the reaction rate is lower.

Alan Boyd's answer covers the mechanistic aspects quite well, but there is another aspect he didn't quite touch on - what's going on at the molecular level. To understand this, you need to think about the actual conditions of the reaction: unless it's taking place inside a cell, which straight biochemical reactions of the kind you're talking about almost never are, then the enzyme, substrate, and product are all floating around at a certain concentration. The enzymatic reaction is proceeding nicely, and as the substrate binding pocket is freed as product dissociates from the enzyme and diffuses away, allowing another substrate molecule to take its place and the next reaction to occur. This is the situation at your 2 mmol [substrate] level. At 50 mmol, the concentration of substrate is much higher, and so even though the actual catalytic portion of the enzymatic reaction is taking place at essentially the same speed, the rate of substrate diffusing into the active pocket is somewhat higher because the local concentration of substrate is higher, allowing for an apparently slightly faster reaction rate.

I think I get what you are saying, but I don't understand your concept of "fairly low local concentration". Concentration is an intensive property of a system, it is what it is.

@AlanBoyd - I reworded it a bit and took out the somewhat confusing reference to local at the beginning. As I was beginning to write it I was going to try to explain a certain phenomenon of some reactions (very high substrate concentrations can actually lead to a slowdown of the reaction as substrate is produced faster than it can diffuse away, inhibiting the enzyme), then I realized the question wasn't really about that and changed the direction I was going, but not all of the wording. Thanks for catching that!

that's it! I think this is the answer I was looking for. Thanks.

In terms of Michaelis-Menten kinetics, the rate never reaches the maximum rate:
$v = V_{max} \times \frac{S}{K_m + S}$
where $S$ is substrate concentration. Notice that however large $S$ is, the term on the bottom line ($K_m + S$) will be larger than $S$, so $v$ will be less than $V_{max}$.
You can think about this in terms of the binding equilibrium between the enzyme and the substrate:
$E + S \leftrightarrow ES$
Since it is an equilibrium, it can never lie completely over to the right hand side.
This is why $V_{max}$ is always determined by extrapolation. In practice of course, at very high substrate:enzyme concentrations your measurements would have to be very accurate to see the difference: if $S$ is $100 \times K_m$ you are looking at a 1% difference. In your hypothetical example you don't mention what the value of $K_m$ is.

Didn't get the last part, 'In practice of course, at very high substrate:enzyme concentrations your measurements would have to be very accurate to see the difference: if S is 100×Km you are looking at a 1% difference. In your hypothetical example you don't mention what the value of Km is.'

@Tytoalba the absolute value of Km doesn't matter because I am simply replacing S with (100*Km) in the 1st equation. My last sentence is just to say that the OP gives values of S but not Km so we can't judge where we are on the curve.


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What are the functions and differences between axons and dendrites?
My textbook doesn't do a very good job of pointing out what the differences between the two are. It basically mentions axons only in the same breath as the synapse (that synapses are the endings/tips of axons).

how about some other textbook, wiki, google? how is this question OK for Bio@SE?

This reference is a bit basic, but lists the functions and differences between axons and dendrites.  Specifically, dendrites receive signals from other neurons, to the cell body; whereas, axons take signals away from the cell body (essentially 'input-output').  A diagram of the parts and the processes is below:

(Image source with additional information)
This Youtube tutorial is a nice visual description of both, and how they function within a neuron.

Can you provide some details in your answer rather than just links?

@kmm is this acceptable?

no synapse.. hmm

@Lesbihonest The synapses are just not labelled on the diagram, but are referred to in the links.

Dendrites

Means Trees in Greek;
Are the input of the neuron;
Receive information from other neurons or the external environment;
Transfer information to the cell body and axons;
Are numerous, relatively short, and branch extensively in a tree-like fashion
May have numerous spines on them to provide a greater surface area for other neurons to synapse on;
Receive information from other cells at these synapses. This makes dendrites postsynaptic.
The connection between axons that synapse on dendrites is called axodendritic;
The connection between dendrites that synapse on other dendrites is called dendrodendritic.

Axon
 - Means axis in Greek

The output of the neuron;
Transfers information to other neurons;
Begins at the axon hillock, which is a swelling at the junction of the axon and soma where there are many Na+ channels and the action potential starts;
Is relatively long (some reaching several feet);
Has terminal boutons at the end where the synapse is located. This makes axons presynaptic. These swellings at the terminal bouton is where the neuron synapses with another neuron;
Contains numerous vesicles which hold neurotransmitter;
Has many Ca2+ channels in the membrane;
The space between the terminal boutons and the next cell is known as the synaptic cleft, and is approximately 20 nm thick;
Most are myelinated, i.e., have myelin sheaths that are made by Schwann cells or oligodendrocytes. Myelin acts as insulator to help conduction of action potential. There are openings between the Schwann cells called Nodes of Ranvier. These help with the conduction of action potentials.
Synapse on other cells in various forms;
Axoaxonal: Axon is connected to another neuron’s axon;
Axodendritic: Axon is connected to another neuron’s dendrites
Axosomatic: Axon is connected directly to another neuron’s soma
In neuromuscular junctions, axons synapse directly on muscles.

Presynaptic and postsynaptic neuron. The synapse is shown enlarged in the inset. Source: Rowland Hall.


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Does osmosis occur when we have two different substances?
Assume that we have a semi-permeable membrane with water on both sides. First, straightforward, case: On the right side, we have a concentration of the substance A, and on the left side, we have pure water. Obvious effect: water flows from left to right.
Now, imagine we have a second case. We have the same membrane, and a water solution of A to the right. To the left, we have a water solution of a different substance, let's call it B. Now, what happens? I can imagine a few possibilities:

the system behaves exactly as in the first case, because the substances have no influence on each other
the system behaves as if it was a solution of A on both sides, with water flowing in the direction of higher concentration (because it only matters if there is something dispersed between the water molecules, but it does not matter what)
some interesting cross-influence effect happens
either one of the three things above can happen, depending on the particular combination of substances A and B

Which one is the true effect? What happens, and why?
If you need an example of the actual substances, let B be thiamine and A be plain sodium chloride, but I am interested in the general case.

If A and B are at the same concentration there will be no net movement of water.
Everything is down to diffusion. In your example of solution A/pure water, water molecules on both sides of the membrane are diffusing through the membrane to the other compartment. However the "concentration" of water is less in solution A, so the rate of movement is less from solution A to water than it is from water to solution A. Water will continue to move into the solution until another force, such as hydrostatic pressure, counteracts the "osmotic pressure".
If solution A and solution B have solute at the same concentration then the rate of movement of water will be the same in both directions, so no net movement will occur.
(In a typical experimental set up using dialysis membrane none of this would apply with the solutes that you mention since they would also be able to move through the pores and you would end up with perfect mixing. You would have to use macromolecular solutes.)

To make sure I understand your answer: you are saying that if we have a high concentration of B on the right, and low concentration of A on the left, water will flow from left to right?

Yes, that's correct.

If one solution can permeate and the other can't, would there be any net movement of particles?

It's not actually diffusion. From Wiki (with source), "The diffusion model of osmosis is rendered untenable by the fact that osmosis can drive water across a membrane toward a higher concentration of water."

Actually, the so-called osmolarity of a solution is only determined by the concentration and number of dissociating species in the solute, and is irrespective of the chemical composition of the solute. This is an example of what is known as a colligative property in chemistry.
The van't Hoff equation gives the osmotic pressure exhibited by a solution:
P = icRT
where P is the osmotic pressure, i is the coefficient associated with the number of dissociating units in the solute, c is the molar concentration of the solute, R is the universal gas constant, and T is the absolute temperature. A nonionic species, such as glucose, would have a value of 'i' close to one as the molecule does not dissociate in solution, while NaCl in relatively dilute concentrations should have an 'i' value close to 2, as it dissociates into sodium and chloride ions in solution.
Thus in the case of the two solutions on opposite sides of the semipermeable membrane as you described, water would flow from the region of lower osmolarity to the region of higher osmolarity, until the osmotic pressure on both sides of the membrane come to equilibrium (due to the changes in concentration of the solutes by the net water movement).


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What do cells do when they aren't creating proteins?
I've always thought that the majority of the "work" in a cell is protein production, until I read the following.
The Wikipedia article on the central dogma of molecular biology states this:

80% of the human genome is transcribed even though only 1% codes for
  proteins.

and this:

Current research focuses on investigating the function of non-coding
  RNA, that is, RNA that does not follow the dogma trend and does not
  code for polypeptides.

If 99% of what's transcribed isn't used for creating proteins, what is it used for?  If it's not known - are there any hypotheses?

in short, some of it is regulatory, like miRNAs, and a bunch of it we don't really know why...

...and of course, even regulatory RNAs are ultimately concerned with the core activity of "creating proteins".

...and from another angle, red blood cells don't make any proteins.

So what do the Ribosomes do in red blood cells?

Red blood cells lose their ribosomes when they develop from reticulocytes. My comment was only meant to point out that not all cells are typical.

OK - I didn't know that, I'm learning a lot - thanks.

As indicated in the comments there are several non-coding (untranslated) RNAs, that the cell produces. Most of these perform gene regulatory functions.
However, widespread transcription is known to take place throughout the genome (pervasive transcription), but the functions of these RNAs are not clearly understood. It has been suggested that in certain cases the event of transcription, rather than its product, is responsible for chromatin regulation.

Untranslated RNA is incredibly important. As mentioned it regulates gene control, siRNA or miRNA for example can cut up mRNA preventing it being translated. Others can cause an increase in expression like enhancer RNA. Then RNA can act as tRNA or ribosomal RNA in protein translation. RNA can be enzymatically active, RNA also acts as a primer for initiation of replication. Then theres retroviruses that have inserted into our genome but have lost the ability to exit and are now part of our genome: the endogenous viruses. There are genes that are redundant duplications of functional genes that are free to evolve. As mutations occur silent parts may regain the ability to be transcribed.

When cells are not producing proteins they are performing their specialized functions.  Consider red blood cells carrying oxygen.  Or gut epithelial cells aiding in your digestion.  The list goes on...liver cells have a different role than the keratin cells in your hair.  There is a lot of transport that goes on as well in an certain cells that is carried out by motor proteins such as myosins, kinesins, and dyneins.  Cells are producing ATP to power these motors and many other integral functions. Also, consider cell replication and the machinery needed for that to occur.
You may visit Wikipedia for more information on motor proteins.
https://en.wikipedia.org/wiki/Motor_protein


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Finding a template/oligo combination for my first PCR experiment
I'm an information technology engineer. I love biology so I research biological topics and have an interest in PCR. That's why I have decided to create a PCR machine.
Everything is done now and I want to do my first experiment to see if it works or not. I would be very happy if someone could give me any suggestions for testing it at home. Which oligo should I use for testing in the easiest way? I mean a template DNA for testing that is easy to find and extract.

You need a genome that isn't too complex, which suggests something microbial. You also need a genome that is well-characterised : either fully- sequenced, or with lots of sequenced genes to allow you to design primers. And finally you need a readily accessible source of material for DNA extraction.
I would suggest either dried yeast, as a source of Saccharomyces cerevisiae DNA, or live yoghurt as a source of DNA from Lactobacilli. You could even try culturing something from yoghurt to get pure bacterial colonies: since these would be from a food source there should be minimal risk.
You might be tempted by one of the traditional plant sources of DNA (e.g. onions) , but it can be tricky to avoid copurifying polysaccharides which inhibit DNA-related enzymes.
Finally, if you do culture some bacteria, or even if you use dried yeast, you probably wouldn't need to actually prepare DNA at all. PCR direct from bacterial cells is widely used - the 95 C step will release sufficient DNA.
added later in response to comment by OP
I chose to follow up on Lactobacillus delbrueckii subsp. bulgaricus because I found this coming up over and over again with reference to yoghurt. If you were to test a range of yoghurts I think you could be fairly confident that this would be present.
There are 5 published genomes for different strains of the species Lactobacillus delbrueckii including subspecies bulgaricus (various strains) and lactis. How different are they? I decided to look at one gene, and lactate dehydrogenase seemed like a logical choice (it produces the lactic acid).
The complete nucleotide sequence for the lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 is here. If you run a BLAST from that page limiting the search to Lactobacilli (taxid 1578) you do indeed find hits to the gene from several other genomes. Here is a representative selection from the sequence alignment to the least similar of the hits:
Query  99766   CCTTCCACGCTTCTGGAGGTACTGAATTCGCCCTGGCTCAACTGGGCAACGATGCCGGTA  99825
               ||||||||||||||| ||||||||||||||||||||| ||||||||||||||||||||||
Sbjct  90616   CCTTCCACGCTTCTGAAGGTACTGAATTCGCCCTGGCCCAACTGGGCAACGATGCCGGTA  90675

Query  99826   TGTACGGTGCCGTTAAGATGGTTCTGTAATTTTGAATGTAAAAAAGCACCAGGCTTTTAA  99885
               ||||||||||||||||||||||||||||||||||||| |||||||||||||| |||||||
Sbjct  90676   TGTACGGTGCCGTTAAGATGGTTCTGTAATTTTGAATATAAAAAAGCACCAGACTTTTAA  90735

Query  99886   AGTCTGGTGCtttttttGTTTATCCTAAAGAGTCCAGGGTTGCCTTTATCGTCGCGGCTG  99945
               |||||||||||| |||||||||| |||||||||||||||||||||| |||||||||||||
Sbjct  90736   AGTCTGGTGCTTATTTTGTTTATTCTAAAGAGTCCAGGGTTGCCTTCATCGTCGCGGCTG  90795

Query  99946   AGGCCCGCATCTTGGCCAATTCGCTGTCATTTAACGGCAATTCCAGTACGTGGCTGATCC  100005
               ||||| |||||||||||||||||||||||||||||||||||||||| ||||||||||| |
Sbjct  90796   AGGCCTGCATCTTGGCCAATTCGCTGTCATTTAACGGCAATTCCAGCACGTGGCTGATTC  90855

Query  100006  CTTGTCCGTTGATGATGGCCAGGGTGCCCAGGTAGATCTCATCCTTGATCCCGTATTCCC  100065
               |||| |||||||||||||||||||||||||| ||||||||||||||||||||||||||||
Sbjct  90856   CTTGCCCGTTGATGATGGCCAGGGTGCCCAGATAGATCTCATCCTTGATCCCGTATTCCC  90915

Query  100066  CGTGCAATGGTGCGGAAAGAGGCAGGGCCAGGTCGTTGTTTTCCAAAATGGCCGTCACGA  100125
               |||||||||| ||||||||||||||||||||||||||||||||||||||||||| |||||
Sbjct  90916   CGTGCAATGGCGCGGAAAGAGGCAGGGCCAGGTCGTTGTTTTCCAAAATGGCCGCCACGA  90975

Query  100126  TCTTGGCCAGCATCATGGCCACGCCATAGTAGGTTGCGCCCTTTTTGACGATAATCTCGC  100185
               |||||||||||||||||||||||||||||||||| |||||||||||| |||| |||||||
Sbjct  90976   TCTTGGCCAGCATCATGGCCACGCCATAGTAGGTCGCGCCCTTTTTGCCGATGATCTCGC  91035

As you can see, there are some differences, but there should be no problem designing primers matching both (again - this is just one part of the gene). Obviously you might wish to make an alignment for all 5 sequences, but I got the impression that at least some of the others were identical to the query, even though they are from different subspecies. These are very closely-related organisms.
In terms of your experiment, I suggest that you plan to use a range of yoghurts and/or starters as the source of template. You could even mix them, if there are constraints on your set-up.

Hello, thank you so much. Live yoghurt is a good idea. So i need to culture Lactobacilli from yoghurt to get pure bacterial colonies before PCR or i just need to PCR direct from live yoghurt ?

I would worry that something in the yoghurt might inhibit PCR. Do you have a centrifuge? Maybe try diluting some yoghurt and then spinning out a cell pellet. This looks like the starting point.

Or you could buy a yoghurt starter culture -might be cleaner.

I can not sure what i buy is exactly same type of Lactobacilli because the seller don't know the scientific name of the bacterial. There are dozens type of Lactobacilli so i worry i need to design a lot of primers.

"Do you have a centrifuge? Maybe try diluting some yoghurt and then spinning out a cell pellet" can you describe more about this method, i never using a centrifuge before but i can create one. How do i know where bacterial located in the tube after centrifuge (is it usually in bottom ?) and how to calculate the RPM suitable ?

@Đức UltraSoft Probably better to ask a new question at this point.

Nice project. How does your machine compare with this open source thermocycler?
Since you are probably ordering a couple of oligonucleotide DNA (primers), I would order a couple more DNA oligos that will work as your template.
Something like this:
oligo 1, primer forward. 5'-AAAAAAAAAAA-3'->
oligo 2, template        3'-TTTTTTTTTTTNNNNNNN[...]NNNNNNNNNNCCCCCCCCCC-5'
oligo 3, template        5'-AAAAAAAAAAANNNNNNN[...]NNNNNNNNNNGGGGGGGGGG-3'
oligo 4, primer reverse                                <--3'-CCCCCCCCCC-5'

This is just to give you the idea, the actual sequences should be longer, have similar GC% content, and similar t-melting. The advantage is that you reduce complexity and you are virtually void of PCR inhibitors.
You will also need some DNA polymerase. Perhaps you can borrow an aliquot from someone working in a biology lab.

Well, amazing idea ! But 4 oligo could make more cost than 2 oligo because i need to ship oligo from foreign country so price is an important factor in my experiment. Anyway thank you for that idea. I'm finding another way simpler than that when DNA can be found in any living organism.

I think my PCR machine is cheaper, faster, special because i using a motor to bring the tube from 95 o C to 65 o C just in a few second. If you like my PCR, i wil sell it for you cheaper than the OpenPCR ;)

I edited my answer: you can do PCR with two oligo without any other DNA template. This is the simplest way I think.

@Alan, thanks I removed from the answer. Fill in, will not be useful to test the thermocycler.

Hello, why you removed the edited answer ? It's look like a good idea, can it's possible if i using only 2 oligo 3'-NNNNNNNNNNNNNNNNAAAAAA-5' and 5'-TTTTTTTTNNNNNNNNNNNNNNNN-3' ?

yeah it can be done using two oligos.. but it is better to not have low complexity sequences like AAAAA and TTTTT etc.

Using only two self-annealing oligos as both primers and template, you will just fill the in withouth amplification. The reaction will occurr even withouth thermal cycling, which renders the presence of thermocycler unuseful. Perhaps the worst experiment ever to do to test a thermocycler :-)  Thanks @Alan Boyd  for pointing it out.   Btw, oligos are normally shipped lyophilized at room temperature in small tubes inside normal envelopes, ordering 2 or 4 will not affect shipping costs.

You are anyways going to get primers and enzymes from somewhere.
The best option would be to get a plasmid from any biolab. Most of the plasmids have specific sequences like M13 promoter sequence, T7 terminator/promoter sequence etc. These are standard sequences derived form viruses, that are commonly used for primer binding sites for sequencing. Also, these primers are pretty standard and optimized for easy PCR and you can get them quite easily.

I agree with this entirely, but the OP said: "a template DNA for testing that is easy to find and extract" (my emphasis)


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How can I create a microcapillary for manipulation of single cells?
i'm working as a diy bio. I'm finding a way to create a micro glass capillary for picking up single cells. I see this video on youtube and would like to know what is the minimum I/O diameter if I pull a capillary like this.
Can it be 10-50 µm at least ?

What kind of cells do you want to pick up? From what sample? Do you absolutely need single cells?

I mean what is the minimum I/O diameter if i pull a glass capillary like that video. I'm working on single cell, inject sperm to egg ...

Well, depends what type of capillary you use (there are many different ones), how close you go to the Bunsen, how fast you pull, how long you leave the capillary on the fire etc. etc. The best thing would be to invest in a pipette puller, it is very difficult to establish. Also, for microinjection you will also need a micromanipulator (unless you have an incredibly steady hand).

well, i'm an IT engineer so i would like to create a micromanipulator. I don't want to purchase for a puller because it will cost too much. I just want to finding a simply way to have one with minimum cost as possible. I wonder that if i pull by hand with fastest speed i will have a capillary with 10-50 µm diameter at least.

Unless you are a skilled machinist (or know one), it's going to be very difficult to make a micromanipulator from scratch.

It is very difficult to establish what kind of tip you will get from pulling a capillary on a Bunsen burner.
There are many variables to consider, including the glass you are using, the temperature you reach before pulling, the pulling strenght, speed and time, whether you pull once or make multiple pulls etc.
If you do not want to invest into a micropipette puller (they are indeed VERY expensive) you could at least build some mechanism to obtain a constant pull in terms of time and force.
The following paper may be of help:
An Instrument for Controlled, Automated Production of Micrometer Scale Fused Silica Pipettes
It seems you would need a fairly high speed (0.8-1 m/s) to get a tip size of <50um.
I could not really find a specific reference for pulling over a Bunsen.

Here is a reference for pulling quartz glass pipettes using a custom oxy hydrogen burner.  Here is a video of a commercial burner based pipette puller.  Not sure what the specs are.  I guess if you can melt your glass using a filament it could be easier than using a torch (a lightbulb circuit is easier to control than flame).  People move to flame when dealing with high melting point quartz.
You can get a micropipette puller from Sutter for $1500 off of Ebay.  I'm not completely sure how they work mechanically, but I have used many models.  Someday I'd like to look inside them (the true mechanism is underneath, inside the box).  They use a metal "boxcar shaped" filament that fits around the cappilary, and this heats up when a current is passed through.  Two pulleys pull at the pipette, attached to something below doing the actual pulling.
I think there might be a spring-loaded mechanism for achieving the pull, rather than a motorized linear stage moving so fast.  Maybe a stage or other actuator below pre-stretches a spring so that it is pulled to different degrees during melt cycle?

Its actually not that hard however its tough to make them consistently my hand. I have a capillary puller in my lab which can give me +-.5um ID we use for a patch clamp setup but When I do it by hand [just like the video] I can get within 10-50um ID. Usually it takes two pulls first to get the elongated cone shape and the second to pull it so thin that it looks like a wire and the orifice has closed. However, if you hook up the pulled capillary via flexible plastic tubing to a syringe you can try to VERY SLOWLY get a droplet and you'll know you're good. Imaging the tip is also a challenge sometimes. There's a wired lens effect with the borosilicate I'm using


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What insect is this? (Black body two orange lines in its back and six legs)
I found this today in the library while studying. I am very curious to know whether this particular insect has a name that I can look up and find more information about:

Full length is almost one centimeter. It doesn't bite or anything. It has lots of small hairs in its back (similar to cockroaches and ants) and has a tail that a white sticky substance comes out from.

where in the world are you?

That is awesome. I have them all over a very large potted flower I have on my front porch. Which also have a lot of aphid on it so assume that is why they are there., ( to eat the aphid ) I do know ladybugs love to eat aphid so this is good as I do not like to put pesticides on my plants or gardens. And I live on the eastern side of Washington State which is very dry and has lots of little critters all over your gardens. Thanks for the info.

Possible duplicate of What is this yellowish insect?

It's a larvae from a ladybird (or ladybug).  Judging by the stripe pattern it is a Common Spotted Ladybird (wiki: Harmonia conformis) and from the body shape & size I'd also say 3rd instar.
The one you have photographed, and the one on flickr, are larval forms of the ladybug, just like when a catepillar becomes a butterfly, the ladybugs also have a larval stage in their life cycle which crawl around and look very different to the adult form.

It is definitely this one: http://www.flickr.com/photos/burtonandy/4628261516/ I just cant understand why the photo of the insect in wikipedia is totally different with this one!

@dane The one you have photographed and the one on flickr are larval forms of the ladybug, just like when a catepillar becomes a butterfly, the ladybugs also have larvae which crawl around and look very different to the adult form. http://www.enchantedlearning.com/subjects/insects/Ladybug.shtml

Wow. I've seen these around a bunch, but I had no clue that they were ladybug larvae. That's wild!


text:
What are the functions of magnetic bacteria?
I'm trying to understand why are bacteria "equipped" with magnetosomes ("intracellular organelles in magnetotactic bacteria that allow them to sense and align themselves along a magnetic field") and why and for what do they need such organelles.

You can find a detailed discussion of this topic here.
Magnetic bacteria contain chains of magnetic crystals (magnetite or greigite) which cause the cells to be oriented in a magnetic field.
It was originally proposed that magnetic bacteria use their magnetosomes to ensure that they swim downwards into the sediment (everywhere on earth, except at the equator, these lines of force have a downward component vector.) However, the current view is more complex than this: magnetic bacteria are microaerophilic and occur in stratified aquatic environments (i.e. ones where there are zones of oxygen concentration at different depths). In this view the magnetic "sense" is not a true taxis, but rather is used to keep the cells oriented in the magnetic field while they adjust their position by sensing oxygen (aerotaxis). As you may be aware, the standard way that Gram-negative bacteria operate is to perform a biassed random walk in three dimensions. The alignment of cells with the magetic field will reduce the dimensionality of the movement, which is more efficient.


text:
What biological constraints are there on how large a spider can grow?
Spiders fascinate me, but what intrigues me the most is just how big some spiders can get, which leads me to my question. What biological constraints (if any) would limit how large a spider could grow, given ample food and no predators?

If predators are killing spiders for food, rather than killing in general, I'd say the primary limit of spider size is how long it take for my wife to spot them!  They don't get very large!

The standard view on this is that the size of terrestrial arthropods like insects and spiders  is limited by the atmospheric concentration of oxygen. This is because they rely upon diffusuion of oxygen into the 'blood' or haemolymph via a system of tubes called trachea that open on to the body surface at the spiracles. As the body grows larger the proportion that has to be taken up by the trachea gets larger. Consistent with this is the presence of fossil giant dragonflies, Meganeura dating from the Carboniferous era when oxygen levels were at an all time high. However you will find that if you go to the Wikipedia page for Meganeura that this idea has been disputed, so the answer may be that no-one really knows.
In relation to alternative explanations; a recent paper by Clapham & Karr (2012) compares fossil insect wing lengths and oxygen concentrations to study the historical drivers of evolution of body size. They conclude that oxygen seems to be the main driver until the end of the Jurassic, but after that biotic interactions (such as bird predation) could be more important.

That is one big dragonfly! Beautiful looking creature.  I wonder if there is any evidence of giant spiders during the Carboniferous.

@Alanboyd We recently had a seminar on a relevant paper, which I added to your answer. Hope you are ok with this.

+1 and accept, thank you for the answer Alan Boyd and for the excellent addition, fileunderwater.  I will ready that paper and post any questions that may arise from it.

One way spiders could become much larger is if oxygen levels in the atmosphere increased. This happened with almost all land arthropods during the Carboniferous period, when oxygen levels in the atmosphere shot up to 35%, as opposed to a measly 20% today. In pure or near near pure oxygen, bugs, including spiders would reach truly monstrous sizes, and by monstrous, I mean much bigger than you, creatures like plane sized flies, tank sized beetles, spiders the size of houses, etc. This is because of the design of the respiratory system in arthropods, as they rely on a series of tubes called spiracles or organs that are similar to gills, known as book lungs, that work in air. These systems rely on diffusion, and this means there is a cap to the size that a land arthropod can grow to, but this cap moves with the oxygen level, the higher the oxygen level, the bigger the land arthropod can get. So yes, there is a way to make giant spiders.

Welcome to Biology Stack Exchange. Please take the [tour] and visit the [help] for more information on this site and how it works. In particular pay attention to the section in the help about what makes a good answer. We particularly value answers with credible references, which yours lacks, and you've made some pretty extraordinary claims about size limits; so lets see some evidence of that.


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Selective Androgen Receptor Agonist
I'm looking for an inducer that strongly activates the androgen receptor, but not the glucocorticoid receptor that is not DEA regulated. I know that SARMS (Selective Androgen Receptor Modulators) are something of a hot topic in some corners of some fields, but I'm having some trouble in my laboratory finding one. My research necessitates the independent induction of AR (Androgen Receptor). Does anyone know how to go about this search or know of some chemical?

Are you looking for literature? If so this is a recent review: Zhang et al. (2013) Deciphering the selective androgen receptor modulators paradigm. EXPERT OPINION ON DRUG DISCOVERY 8: 191-218   DOI: 10.1517/17460441.2013.741582

Take a look at the paper by Ojasoo et al. [1] It reports the relative binding affinity for a wide variety of steroids for the AR, PR and GR.
Nandrolone (19-nor testosterone) does not have any affinity for the GR, but is a potent activator of the AR. However, it does have some affinity for the progesterone receptor (does this matter for your research?). Additionally, nandrolone bound to the AR also binds estrogen response elements (does this matter for your research?).
5a-DHT also is a potent AR activator with no affinity for the GR. Do note that a metabolite of 5a-DHT has significant affinity for the estrogen receptor, might that matter for your research.
You might also use a strong GR antagonist in conjunction with an androgen which also has some affinity for the GR. Then you can simply use methyltrienolone, as commonly used as AR ligand.
On a sidenote: do note that androgens usually only lead to significant dissociation of a GR ligand for the GR at very high concentrations. Therefore, androgens most likely act as GR antagonist by heterodimer formation of the AR with GR [2], as well as through squelching [3].
References

Ojasoo, T., et al. "Towards the mapping of the progesterone and androgen receptors." Journal of steroid biochemistry 27.1 (1987): 255-269.
Chen, Sei-yu, et al. "Androgen and glucocorticoid receptor heterodimer formation A possible mechanism for mutual inhibition of transcriptional activity." Journal of Biological Chemistry 272.22 (1997): 14087-14092.
Zhao, Jingbo, et al. "Oxandrolone blocks glucocorticoid signaling in an androgen receptor-dependent manner." Steroids 69.5 (2004): 357-366.


text:
How does ubiquitin recognize misfolded proteins?
Misfolded proteins are tagged by ubiquitin and then are destroyed by proteasomes. How does ubiquitin know which protein to tag?

http://www.youtube.com/watch?v=hvNJ3yWZQbE I think this video explains the ubiquitin system very well.

Ubiquitin and the UPS
The ubiquitin proteasomal system (UPS) is a common method of regulation for many proteins in the cell. The modification is actually generated by E3 ubiquitin ligase, of which there are ~400-800 in the mammalian cell and the main source of specificity in the UPS (E2 ~40 and only 1 or 2 E1).
Ubiquitin gets its name from ubiquitous, meaning found everywhere. It's a 76 amino acid molecule that modifies proteins at lysine residues. Monoubiquitination can act as a signalling molecule whereas polyubiquitination leads to degradation. Ubiquitin is first activated via an ATP-dependent pathway to create a high energy thioester with E1. This is transthiolated to E2 and finally E3 allows the addition to the target protein. So really, your question is how E3 ubiquitin ligase recognises its target. 1
E3 ligases
There are 3 classes of E3 ligases, according to whether they contain a RING, U-box or HECT domain. These domains are responsible for either directly catalysing the addition of ubiquitin or simply facilitating another E3 ligase to do so. RING and RING-like domains are by far the most common (<600). Exactly how these domains mediate the transmission of ubiquitin is not clear, but it can be via an E3-Ub intermediate or directly from E2 to the target. The domains are relatively standardised, due to the fact they bind to E2 proteins, which contain less diversity than target proteins. However, it's the combination of the E2 and substrate binding domains that generates the selection. 2
Target recognition
So how does the target come into all of this? Each E3 ligase recognises a specific sequence on the target protein due to it's own conformation and signal sequence creating a unique interface full of intermolecular interactions- just like any protein recognises any other protein. However, although individual cases vary, the tight control primarily comes from post translational modifications on the target. Phosphorylation in particular is common in signalling and may allow E3 to bind, inducing degradation of the target. I'll use some examples to demonstrate this point, but note that every case will be different.

c-Cbl (E3) recognises activated receptor tyrosine kinases and ZAP 70/Syk kinases (target) by binding a phosphotyrosine sequence through its tyrosine kinase binding domain (containing SH2 domain). The phosphorylation is controlled by other cellular signals
Glycosylation
Proline hydroxylation
Sumoylation

Other regulations of the process that are linked but perhaps outside the scope of your question include E3 post translational modifications (eg. autoubiquitination), sequestration of E3 via binding partners (Cand1 and Roc1), pseudosubstrates and substrate competition. 7
Misfolded protein degradation
Making protein is a large investment for the cell and so there are several protective measures to prevent misfolding/aggregation. Whilst there are many different types, chaperones generally work by isolating the misfolded protein and undergoing a series of binding release steps, triggered by hydrolysis of ATP. The protein can still be released unfolded, and the cell will continually try to isolate it for refolding. However, certain chaperones form a complex with the proteasome, and can recruit other UPS components. The choice to give up on refolding appears to be a stochastic process, and will depend on the concentration of the appropriate chaperones. Misfolded proteins tend to have significant regions of exposed hydrophobic residues, and E3 ligases are therefore likely to recognise this less specifically than the degradation pathway described above.
Relevant sources include endoplasmic reticulum associated degradation and chaperones.
Summary
The answer to your question is that there is no standard recognition sequence in E3 to know which protein to target, the binding needs to be diverse in order to give the control the cell needs. That means it could be anything, but each E3 is likely to be relatively specific for its target, and is often controlled by other post translation modifications on the substrate. Misfolded proteins can be recognised and degraded due to the association of the UPS with chaperones.
If you're really interested, you can download this file and open it in something like PyMol to actually see the ligase ready to transfer ubiquitin.

Ubiquitin is active as part of a complex of proteins. Other elements of the complex recognise the proteins to be degraded. These recognition or specificity proteins are responsible for targetting proteins for ubiquitination. They are called ubiquitin ligases.

That’s just begging the question. Now OP knows that ubiquitin doesn’t tag the proteins itself but they still don’t know how proteins are tagged, i.e. how E3 recognises the misfolded proteins.


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What Effect would Cannabinoids have on the Acute Porphyrias?
I should be clear in saying that this question is NOT intended for personal medical advice. Rather what I am looking for is journal articles and/or books that touch on this subject because I think it would be quite an intriguing topic to probe. I am unaware of any interaction between the endocannabinoid system and the formation of haem and porphyrins in general.

Do you have reason to suspect they would have an effect?

Sort of, I know they can interact with the P450 cytochromes and hence may bring on an acute attack.

A Web of Science search for endocannabinoid AND porphyria under Topic produces zero hits.

Yes, they can induce an acute attack.

Drugs metabolised by cytochrome P450 (CYP) such as analgesics may induce acute attacks in patients with hepatic porphyrias. In recent years, preclinical and clinical studies have suggested that cannabinoid pharmaceutical preparations may be potentially useful in the treatment of pain [1].

There is even a clinical case about acute intermittent porphyria in a marijuana user:

Mr. G is a 55 year old Caucasian male with a past medical history significant for acute intermittent porphyria (AIP), chronic back pain, renal insufficiency, hypertension, and nicotine dependence. The patient has presented to the hospital with multiple AIP exacerbations since diagnosed eleven year prior to the current case. [...] Urine drug screen was positive for cannabinoids, which he admitted to frequent marijuana use [2].

But an experimental study on mice failed to prove that:

Our findings indicate that CP-55,940 (a cannabinoid CB1 receptor agonist) and its metabolites do not behave as porphyrinogenic drugs and may potentially be safe for treating pain in patients with acute porphyrias [1].

References:

Fontanellas A, Manzanares J, García-Bravo M, Buzaleh AM, Méndez M, Oliva JM, Batlle A, Palomo T, Enríquez de Salamanca R. Effects of repeated administration with CP-55,940, a cannabinoid CB1 receptor agonist on the metabolism of the hepatic heme. Int. J. Biochem. Cell Biol. 2005 Aug;37(8):1620-5. doi: 10.1016/j.biocel.2005.02.010. PubMed PMID: 15896668.
Ware SK. Acute Intermittent Porphyria With Psychiatric Manifestations: A Case Report. Ment Health Clin. 2013;3(5):100. Available at: http://cpnp.org/resource/mhc/2013/11/acute-intermittent-porphyria-psychiatric-manifestations-case-report. Accessed July 27, 2014.

authors: افراح الجهني


text:
Which are the most extreme artificial environments that lifeforms/ecosystems have adapted to?
I have heard of 'extremophiles', lifeforms that naturally thrive in all sorts of extreme environments.
Is there any evidence to suggest that lifeforms are adapting to the extreme environments (such as in mining waste, nuclear waste etc) that result from human activity?

This seems to be two questions. Are you interested in adaptations to extreme environments in general, or do these have to be recent adaptations to man-made conditions. If it is the latter then I don't think you will get any examples since all the "best extremes" have already been around for millions of years. You mention mining as an example, but of course there have been geological extremes since the birth of the planet.

@AlanBoyd good point, with this in mind, I think adaptations to extreme envirinments in general will be more than adequate. These can be inferred to modern environments.

This looks relevant.

@AlanBoyd indeed that is the kind of thing I am interested in.


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Method to recognize the age of a firebug from the pattern on its back
Can I recognize the age of a firebug from the pattern on its back?
If yes, what pattern shows what age/stage?
Below is what I extrapolated from Wikipedia, but is there a more precise scale with more steps? (I could not find more than this with google).
Nymph: Adults:

The answer is more or less yes. Normally Firebugs (Pyrrhocoris apterus) go through 5 nymph instar stages (as do most Hemiptera), where they resemble the adults more and more as they grow. Only adults are winged and have have functional reproductive organs. This type of metamorphosis is called hemimetabolous or simple metamorphosis (in contrast to Holometabolism or complete metamorphosis). However, I cannot find a good overview of the instar stages of Firebugs to link to.
A generic overview of Heteroptera (True Bugs) can bee seen here
(from www.amnh.org/learn/biodiversity_counts):

This picture shows (at least) three instars + adults of Pyrrhocoris apterus (from http://www.projectnoah.org/spottings/17248017). The large nymph with 3 dots along the midline is the final 5th instar nymph:

However, due to hormonal disruptions there have been cases with even more nymph stages (6th and 7th instars with adult characteristics), but this is under unnatural laboratory conditions (e.g. Slama & Williams 1965 and Konopova et al. 2011). Konopova et al (2011) also has pictures of instar stages 4 & 5 (along with gene knowdown versions).

Nice referenced info. The first image would be even better if indicated the patterns too :-)

I'm sure you can find one for Firebugs in a suitable book. However, the graph I posted is a general overview of Heteroptera, which consists of >40000 species, and the growth patterns naturally differs quite alot within the taxon.


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Confusion regarding photosynthesis and respiration in plants
During 24 hours there is a time in i.e. twilight when plants neither give oxygen nor carbon dioxide why is it so?
This also suggests that neither of the two vital processes i.e. respiration or photosynthesis is being carried out.
Well,
during twilight there is not enough sunlight so photosynthesis may  slow down or even cease but what about respiration?

Plants do have mitochondria and therefore their cells undergo respiration.

@leonardo Then after the completion of Kreb's cycle carbon dioxide should be released but during twilight neither is oxygen released nor is carbon dioxide.. and i am not able to understand why..

@AanalDesai do you have a credible reference for this?.. "Then after the completion of Kreb's cycle carbon dioxide should be released but during twilight neither is oxygen released nor is carbon dioxide"

@GruffinEvo7 I am quite sure that during twilight neither oxygen nor carbon dioxide is released but the thought of compensation point just flashesd my mind maybe it could be the reason for no release of oxygen or carbon dioxide....

Plants collect energy (from light, through photosynthesis) to do use of it, just like any other living system do. To use that energy, plants require to do respiration.

@User3800 its very justified that you confused at the stoichiometry of the 2 reactions (photosynthesis and respiration). Actually its the speed and amount of these reaction, (not the stoichiometry), that determines accumulations and equilibria.

You haven't directed us to any evidence for your assertion, so it is difficult to evaluate.
I'm not a plant physiologist, so I will argue from first principles:
photosynthesis consumes CO2 and produces O2:
6CO2 +6H2O -> C6H12O6 + 6O2
respiration consumes O2 and produces CO2:
C6H12O6 + 6O2 -> 6CO2 +6H2O
Plants do both of these things at the same time for at least part of the day. During the day, in a growing plant, photosynthesis will exceed respiration: a lot of the fixed carbon is fed into anabolic processes to produce "plant stuff"; polysaccharides, proteins, nucleic acids. This means that the plant will be a net producer of O2 and a net consumer of CO2. At night there is, of course, no photosynthesis and respiration will dominate and the plant will be a net producer of CO2 and a net consumer of O2.
At certain times of the day, twilight for example, there will be a transition between these two states, and the plant will pass through a state where there will be no net flux in either of the gases.

Well, Could it be due to compensation point that neither oxygen nor carbon dioxide is released?

Just looked at "compensation point" on Wikipedia. Yes that is exactly what I had deduced must happen. Like I said I'm not a plant phyiologist. Was your question meant to go beyond this idea?

No, it answered my question.


text:
Can a dNTP be built into a RNA strand?
DNA consists of deoxyribonucleotides, RNA consists of ribonucleotides. They differ mainly (apart from the uracil / thymine difference) in the sugar part, the deoxyribose and the ribose. Those two molecules differ in the hydroxy group in the ribose which is only a single proton in the deoxyribose. This part of the sugar molecule is not directly involved in binding reactions, nevertheless it causes the whole difference in RNA and DNA.
I wonder: could a dNTP be used in an RNA strand (or vice versa)? Is it chemically possible that we have a RNA molecule that contains a dNTP next to its NTPs?

Are you interested in whether this happens naturally, or if we can do it in the lab? And depending on which interactions you mean, it's not really correct to say that the 2'-OH is not involved in binding.

I'm interested in both -- if we can do it in the lab, it's technically feasible and this would be interesting. But if it is possible, could it even happen naturally? And you are of course right, the 2'-OH is involved in binding, I tried to say that it is not used to form a new bond (therefore not directly involved).

This is rather easy to do if you synthesize oligonucleotides chemically and not enzymatically. This is typically done using phosphoramidite chemistry, and it allows for the synthesis of chimeric RNA/DNA oligos. You can even incorporate modified nucleosides like 2'-O-Me or LNA.
This is typically done if you want to change the properties of an oligo, e.g. if you want to make it resistant to degradation by enzymes.

That plus the paper Alan Boyd posted below answered my question. Thank you!

All DNA polymerases use an RNA primer during DNA replication - they cannot begin a DNA molecule de novo. Before the primer is removed the newly synthesised strand is an RNA-DNA hybrid. The first deoxynucleotide to be incorporated into the new DNA is added to the 3'-OH of an RNA.
Does this count?
Added later:
this looks like a place to start reading

It counts. But I still wonder if it is possible to find a deoxynucleotide in the middle of an RNA strand. BTW: thank you for correcting the question :-)


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Can PCR tubes be made of different materials than plastic?
Does it matter if I replace the PCR tube (usually made of plastic) with another material like aluminum, glass, or something similar?

for someone with time to read & create an answer, here is a paper about glass pcr tubes http://www.biotechniques.com/BiotechniquesJournal/2008/April/Plastic-versus-glass-capillaries-for-rapid-cycle-PCR/biotechniques-44804.html

The key property would seem to be, at first glance, thermal conductivity. According to this table copper wins. But then, what about chemical reactivity? Maybe gold is a better choice. And that brings us to cost and disposability. These factors make the choice of a metal problematic. That leaves plastic and glass. Glass has better thermal conductivity, but clearly it has other drawbacks.

Next to the cost for aluminium or any other metal, a lot of metal ions have chemical functions that makes them not suitable to do chemical reactions in tubes of that material. Copper, for example, reacts with oxygen and can therefore affect the reaction.
The article that rg255 posted in a comment says clearly that glass tubes have advantages compared to plastic tubes:

Heat transfer into plastic capillaries was slowed by thicker walls, lower thermal conductivity, and a lower surface area—to-volume ratio than glass capillaries. Whereas the denaturation and annealing target temperatures were reached by samples in glass capillaries, samples in plastic capillaries fell short of these target temperatures by 6°–7°C.

Nevertheless glass might have a big disadvantage: it breaks much easier than plastic. When you handle the tubes in the lab and don't pay enough attention you might lose your samples, and if you want to freeze some tubes and they are pretty full the glass might break due to the bigger volume of the frozen probe.


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Carotenoid biosynthesis in yeast
Does Budding Yeast Sacchromyces cerevisiae produces significant amount of carotenoids?
Have anybody estimated the ratio of flux going in branches 1. Cholesterol synthesis(via squalene)
2. Coenzyme Q6 and 3. Dolichols Glycoprotein and others from the branch point of Farnesyl diphosphate.
Here is the kegg pathway
http://www.genome.jp/kegg-bin/show_pathway?map00900
I also have pathway formatted but couldn't upload.

The enzyme phytoene synthase is the first committed step in carotenoid biosynthesis. Saccharomyces cerevisiae doesn't have this enzyme, nor does it have the rest of the relevant pathway. Other yeasts such as Rhodotorula spp and Phaffia rhodozyma can make carotenoids.
However, carotenoid biosynthesis has been engineered into S. cerevisiae.

what about the distribution of fluxex from Farnesyl


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How different is an introverts brain different from an extroverts?
How is an introverts brain wired differently than an extroverts? Just to give you an example. On an average, the processing that goes on in an introverts brain is greater than in an extroverts [citation needed].
What part of the brain affects personality traits such how introverted someone is? How is this part of the brain different in either it's physical entity or it's activity between extroverts and introverts?

I think we are several years away from understanding how those type of behaviours are coded in the brain...

This question might fare better on cogsci.SE.

I think you will have a better chance of getting an answer if you narrow down the question and include references for your claims.

i think you can look at an extreme case. It's called Williams-Beuren Syndrome. The patients have a deletion in 7th chromosome (q11.23) and have a highly naive and sociable behavior [there are also other markers, of course].

Do introverts have "brains"?  I think this is actually a controversial subject.  Certainly, some have highly connected networks that are somewhat analogous to a brain, but I think of brain as a uniquely vertebrate organ.

I am not sure if this answers your question, but according to the article "The Difference Between Introverts and Extroverts" (Gallagher, 1999), the main differences between introverts and extraverts are where the main brain activity occurs (as seen by positron emission tomography (PET) measurements).  The full PET study is in the article "Cerebral Blood Flow and Personality: A Positron Emission Tomography Study " (Johnson et al. 1999).
The conclusion of the Johnson et al. paper states:

This study revealed a pattern of increased blood flow in the frontal lobes associated with introversion. ... in the present study, there were more cortical regions associated with introversion than with extraversion. ...in the present study, extraverts did show lower blood flow in several regions in the behavioral inhibition system, namely, the frontal lobes and the hippocampus.

Some images of brain activity is included in a more recent article "Differences in regional brain volume related to the extraversion–introversion dimension—A voxel based morphometry study" (Forsman et al. 2012).  Forsman's article looks at activity in  the white and grey matter of the brain.
I hope this helps.


text:
Why do we squint when tasting very sour things?
Sometimes while tasting a very sour thing (like tamarind, lemon etc.) our eyes squint immediately and involuntarily for a second, but a little later becomes normal again.
Why, and how, does this reflexive movement occur?

Maybe it serves to show others that we may be consuming something poisonous. We cry when we are sad to alert others of our distress. There may be better ways to show something is poisonous, but a child doesn't have any real way to alert who ever is feeding it that it may be poisonous. If I was feeding my baby something, and they had that reaction, I would not give that to them again.

This is basically a "just-so story", not a real explanation.

Short answer
Adverse stimuli in general are accompanied by contraction of facial muscles. It is part of our repertoire of emotional expressions.
Background
A disgusted expression and the expression of pain are both accompanied by squinting. Disgusting stimuli include your example of a sour taste, but also include other adverse tastes (bitter) or visual stimuli such as a gross-looking objects. In other words, squinting is likely a common way of expressing adverse stimuli. So the "sour look" may not be easily explained on its own. Instead, it is part of a huge emotional expressive repertoire that makes us human beings. To be able to read these expressions allows us to be empathetic towards others. People with autism and Asperger's syndrome may not be able to do so and are disadvantaged in their social behaviors.
To illustrate the commonalities between sour (#1), disgust (#2) and pain (#3) I would like to share the following web finds.

Sour taste. Source: Getty Images
!
Disgust. Source: Berkeley Emotional IQ test

Pain. Source: Berkeley Emotional IQ test
PS: I am not a psychologist - Consider my contribution as an educated guess.

Squinting is the organism's way to warn us that we are eating something that could eventually be dangerous, acidic or toxic. It's a reflex, developed many many years ago when the ancient people did not know much about the safety of plants, roots and other foods. They used to try them and decide if the object is eatable or not according to its taste.
Also, our tongue has very sensitive papilla, which react to sourness and make the muscles contract.

Do you have a reference, by any chance?

No, actually this particular question was brought during biology lessons a few years ago and I just remember my teacher's explanation.

This is completely unsubstantiated. There was a (now deleted) answer by @Brendon that makes much more sense: fungiform papillae are innervated by the VIIth cranial nerve. Squinting is most probably a side effect of the activation of this nerve. Whether there is an evolutionary advantage to it is debatable, although for sure there does not seem to be any obvious disavantage.

@nico If it weren't advantageous I can't see why it would appear in all (or almost all) of the human population - surely it'd be like the ability to roll your tongue?

@AndroidPenguin: I am just saying that I see no proof that it is there because it has an advantage. It could be there just because it has not be selected against, which is a different thing. The answer gives no proof either way.

This doesn't even make sense.  The sensation of a sour taste already serves as a warning.  How would squinting help?


text:
Some question about materials for PCR
I'm planning to order oligo, polymerase, nucleotide, buffer for my first diy PCR experiment (now I have nothing), so I have some questions.

How and how long I can storage them?  I have only a fridge that can freeze to -10 -> -20C.
I see the size/concentration of polymerase is 400 units and 25 nmole of oligo. So what can I do with 25 nmole oligo and 400 units of polymerase? How many PCR experiment I can do with 400 units of polymerase, and how many PCR experiment I can do with 25 nmole of oligo?
If I have a HPLC machine, can I reuse the polymerase by purify it after reaction?

At -20 C, you can keep things around for a pretty long time. I'm not sure that years is a good idea, but at least months.

The manufacturers of each component should have information available on their website regarding storage temperatures, along with recommended protocols on how much of each component to add to the PCR reaction. I would not try to reuse the polymerase, as depending on the kind you buy it may be a complex of proteins, and it may not be functional after purification. However, you're welcome to experiment all you want, it's your stuff!

1: -20 is the normal storage condition for PCR reagents in the lab so you should be ok. One warning: you should make sure that your freezer is NOT auto-defrosting. Most consumer freezers will cycle temperature to prevent frost build up. This is very bad for enzymes and will greatly reduce their lifespan. If you can’t find a non-defrosting freezer, you might be ok if you buffer the temperature by keeping your enzyme in a cooler in the freezer, or inside a block of metal with a hold drilled in it.
2: You can stretch your reagents by lowering the volume of each PCR reaction. 25uL is workable in the lab, but keep in mind that the smaller the volume the larger the relative error in your measurements becomes.
3: It seems strange to me that you have access to a HPLC machine but can’t afford to buy new enzyme when you run out. I really wouldn’t try to reuse anything. Maybe it could work, but would you really trust your results? If you shop around you can find cheaper brands of polymerase.

Good luck!

3: depends on the polymerase: some of the more expensive polymerases will loose some of their functionality (e.g.: "breaks" that avoid accidental polymerization prior initial heating)


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Can PolyA tails be within Expressed Sequence Tags?
Can polyA tails occur within (rather than at the end) of a sequenced tag? Consider, for example the following two sequences from NCBI:
DY008075
>gi|119423037|gb|DY008075.1|DY008075 19ACACYS_UP_022_A11_29OCT2004_095 Brassica napus 19ACACYS Brassica napus cDNA 5', mRNA sequence
TGGTACGGTCAGATGCTTGCTAAAGGAGAAATAAATAGAGACATGGGTGATAGTATAAGCGGAAAGGGAA
TGATTCAGGGTGTTTCTGCAGTGGGAGCGTTTTACCAACTGCTTAGTCAGTCCAGCCTAAGTATATTGCA
TTCTGAAGAGAAGAAACCTGTGGCTCCGGTTGAATCATGTCCTATTTTGAAAACACTCTACAAGATACTC
ATCACAAGAGAACAATCAACACAAGCGATTCTGCAAGCATTAAGGGATGAAACACTGAATGACCCAAGAG
ACAGGATTGAGATTGCACAGAGCCATGCATTCTACAGGCCTTCCCTTCTAGATCAGCCTTGATTAGTCTG
TCATGGCTCATAATCCGAACTTCTAAGATCTTACTTGTGCAAACTGCAGATTCTGCTATGTTAAACATCA
TGTCTTAAAATTGATTGTTGTTCAGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACATGTC

or EE485195
>gi|126492146|gb|EE485195.1|EE485195 DHBN8DCT_UP_012_C05_25FEB2005_043 BRASSICA NAPUS SEEDS BNDH8DCT Brassica napus cDNA 5', mRNA sequence
GTTACAGCTGGTTGAGAACAGTGACAATTCCCGGTTGAGCAAAGAAATTGCAGACAAGAGCCACCAACTA
AGGCAAATGAGAGGAGAGGAACTTCAAGGACTTAACATAGAAGAGCTGCAACAGCTGGAAAAGGCCCTTG
AAGCTGGTTTGACGCGCGTGATTGAAACAAAGAGTGAGAAGATTATGAGTGAGATCAGTGACCTTCAAAG
AAAGGGAATGAAATTGATGGATGAGAACAAGCGGCTAAGGCAGCATGGAACACAACTAACAGAAGAGAAC
GAGCGACTAGGCAAGCAAATATATAATAATATGCATGAAAGATACGGTGGTGTTGAGTCGGAGAAGACCG
CCGTGTACGAGGAAGGGCAGTCGTCAGAGTCCATTACTAACGCCGGAAACTCCACCGGCGCTCCTGTTGA
CTCCGAGAGCTCCGATACCTCTCTTAGGCTCGGCTTACCGTATGGCGGTTAGAGATGGAACCATACAAAG
AAGTTCATGGAGTGAGGAGATGCTCTGTAGTAACAAGTGGCAATGTAGTAATTTCTCTTGTTTGATGTAA
GTTTTTGTCTGAGGAAGAGGTTTTCCTTTTATGTTCTCTTTGATATTATTATCTTTCTTCACTGCAAAAA
AAAAAAAAAAAAAAAAAAAAAAAACATGTC

It seems to me that both the polyA sequences at the end are some sort of tail rather than actual coding for poly lysines. If we BLAST either of the sequences, the polyA part doesn't align with any reliable nucleotide or protein (i.e., with the NCBI non-redundant databases). I can give more examples and show their best alignments to nr-Sequences but it will make the question too long.
Cross posted to SeqAnswers.

As you rightly put, these poly-A sequences are not a part of the gene. I haven't done EST analysis but I generally trim poly-A tails and filter out sequences with more than 7-10 contiguous As, for RNAseq

@WYSIWYG, but I thought they had to be at the very end. Why is there a CATGTC at the end after the polyA tail?

i tried BLASTing the second sequence against Brassica napus nucleotide collection. There is a match but only uptil 472nd residue of the query. The rest of the sequence (473-uptil the beginning of poly-A) didn't have any match in Brassica. (some gapped alignments with other non-plant genomes). Although the entry in NCBI says that the authors have removed the vector and adapter sequences, it seems that it is some kind of contaminating DNA sequence. Check their experimental procedures.

The CATGTC sequence at the end of the poly A tail is an artefact of the method used in constructing the original cDNA library.
According to https://www.ncbi.nlm.nih.gov/nucest/EE485195.1 this EST comes from a library constructed in the Clontech vector pDNR-LIB
The Clontech SMART cDNA cloning system manuals are linked to from here
and the general manual descibes the use of a primer for 1st strand synthesis:

CDS III/3' PCR Primer  5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3'
(N = A, G, C, or T; N-1 = A, G, or C)

If you look carefully at the primer you will see that the oligo dT portion, designed to anneal to the poly-A tail of the mRNA is preceded by the sequence GACATG and a SfiI site that is used in some clever cloning strategy that I don't fully understand. What is clear however is that the use of this primer will put CATGTC immediately after the poly A in the cDNA:
           .....SfiI....
5'-ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTT...
   TAAGATCTCCGGCTCCGCCGGCTGTACAAAAAAAAAAAAAAAAAAA...5'

>>> turning bottom strand around -  5' ...AAAAAAACATGTC

Any idea about filtering diverse conditions on the poly-A tail? Follow up question : http://biology.stackexchange.com/q/9104/3922