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Why aren't we allowed to refreeze previously thawed breastmilk in refrigerator?
https://www.breastfeeding.asn.au/bf-info/breastfeeding-and-work/expressing-and-storing-breastmilk

Previously frozen— thawed in refrigerator but not warmed
4 hours or less (ie the next feeding)
Store in refrigerator
24 hours
Do not refreeze

But in freezer the temperature is still below normal, still is there a probability of bacteria growth?
I wish to understand the reasons behind the warning. Answers with references will be appreciated.

Something to note is that the thaw and refreeze cycle is not just about texture, but there is real damage happening to the proteins that are present in the milk. All the good things that make breast milk useful for infants (growth factors, hormones, antibodies, etc) will be destroyed by freeze-thaws.

This is just a guess, but I don't think this is a food safety issues, its more a case of 'this food is gross now' issue.
When I was a teenager I worked in a frozen food processing plant.  The freezers were giant boxes where freon was condensed on gigantic coils into a liquid and then the liquid freon would rain down on to the food passing under on a conveyer belt.  The vegetables were frozen instantly and it was quite tasty - frozen just a few minutes after being cleaned.
As industrial freezing is very quick and such flash freezing, the quality of the food is preserved in a pretty fresh state. [its horrible for the ozone in the atmosphere but this was in the 80s and I hope they changed the process].
Milk is a mixture of water based nutrients and globs of fat, which give the milk is homogeneous white color and its smooth texture.  Freezing milk in a typical refrigerator would take hours and ice crystals would form and separate out the two phases - the fatty portions would separate.  Try it with a glass of milk and see.   This is the same reason that you don't re-freeze ice cream, which has even heavier creme proportions (even fattier).  The texture is completely gone.

so how would fat separation effect health? Is harmful?

I don't think it would- it might be a bit more like skim milk.  The nutrients are still there and if its been frozen after only a short period there shouldn't be too much bacterial growth, but if you think food should be appetizing to be healthy you might not completely feel its as good as if it hadn't been refrozen.  On a molecular scale there could be some differences; if I was having lactovegans over for dinner I'd throw it out.  If I was starving and waiting for a check to come in I wouldn't think twice about drinking it.

oh breast milk - not sure why I didn't think of this.  We've actually refrozen breast milk and used it. This is not medical advice, but in our experience it wasn't a health hazard.

I think this recommendation is simply to be on the save side in terms of degradation. You don't want to feed babies with milk which is not ok anymore - so you take measures that exclude this for sure. Breast milk is a raw product, so if its not handled with care, it can be contaminated.

i agree @Chris .  if you refreeze how long can you keep the milk between freezings?  what temperature is the bottle?  how clean is it?  too complicated for a public guideline.

When I lived in the UK, most supermarket frozen foods came with a "Do not refreeze after opening pack" warning. This is only anecdotal, but I've never heard of anyone getting sick after ignoring this advice. I myself used to ignore that advice quite regularly.

I don't know why I can't comment, but this seems simple enough that you could do an experiment and freeze it and see what happens. One possibility that comes to mind is that fat globules formed during freezing will clog the nipple on the bottle and make it harder for the baby to drink. Breast milk isn't homogenized, so the fats are more likely to separate than normal cow milk from the store. It also isn't pasteurized, so it will spoil faster than store bought milk too, but if you keep it cold and don't let the experiment run for more than a couple days it should be ok. To be safe, don't feed the experimental sample to the baby.


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What are the ingredients of Pheromone Trap using for controlling Fruit flies of cucumber?

Pheromone Traps are used for fruit fly control. But I have no idea which ingredients or chemicals are used for preparing Pheromone trap

What species do you mean - is it Bactrocera cucurbitae?

I have mention about Bactrocera cucumis

source: Stack Exchange

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text:
Definition of "Niche Complementarity"
What does "Niche Complementarity" mean?

On this website they define "Niche Complementarity" as:

The tendency for coexisting species which occupy a similar position along one niche dimension

I don't quite understand this definition. The tendency to do what?

Notes:

I know what is an ecological niche and therefore what is a niche dimension. There is no need to define this.
I am particularly interested in the use of this concept in speciation and adaptive radiation.

There's more to the definition, it's less clear because of the inserted e.g.'s. The definition is: "The tendency for coexisting species which occupy a similar position along one niche dimension to differ along another;" I took out the examples of altitude and diet.

@A.Kennard I still donn't get how you manage to read the whole definition but anyway, the important thing is that I now understand the concept of "niche complementarity"! Thank you. You can make an answer out of your comment if you wish.

As it has been said by @A.kennard in the comments, the definition the OP found is not complete. The full definition on the link he gave (here) is:
Niche Complementarity:

The tendency for coexisting species which occupy a similar position along one niche dimension to differ along another.

The concept that two competing species will tend to evolve different evological niche is called character displacement. The definition from wikipedia is

Character displacement is the phenomenon where differences among similar species whose distributions overlap geographically are accentuated in regions where the species co-occur, but are minimized or lost where the species’ distributions do not overlap.

There is a really good discussion of this in Chesson & Kuang Nature 2008, they propose a way to measure niche complementarity accounting for competition and predation.

Niche complementarity: If the competitor species overlap for one resource dimension then they will differ for another resource dimension. E.g; All the plant species pollinated by humming birds flower at different time of the year.

Welcome to Biology.SE! Please add some references to your answer.

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text:
What's a replicate line?
The methods section inside a paper I'm reading make mention of replicate lines.
Example: "We founded 10 replicate lines from a single clone". This is in the context of experimental evolution and artificial selection of Chlamydomonas in a laboratory.
Can you please explain to me what a replicate line is?
Here's a reference to the paper:
Collins, S., Sültemeyer, D., & Bell, G. (2006). Rewinding the tape: selection of algae adapted to high $CO_2$ at current and Pleistocene levels of $CO_2$. Evolution 60, 7, 1392-401. DOI: 10.1111/j.0014-3820.2006.tb01218.x

It would certainly be easier to answer if we had a link to this article.

Or even a mention of what experiment they did, some context around the quote, anything really. Please [edit] your question to add more information, in the meantime I am voting to close as unclear.

Good point. I added a reference, and some clarification of the context.

In the context of experimental evolution, replicate lines are simply separate experimental (or control) lines the are established from the founder population at the beginning of the experiment. In this paper, the authors must have established 10 separate experimental lines that then evolved in different carbon dioxide levels. For example, 10 separate aliquots of algae are drawn from the same source population and then separately subjected to the agent of selection. These replicate lines should remain separate and not be mixed or crossed.
Replicate lines are useful in experimental evolution for a few reasons. Because you are studying a population that is experiencing a selection pressure, you expect that genetic changes are in response to that pressure. However, changes can also occur due to drift. Replicate lines (and a consistent response to selection among them) is important to ensure that changes are a response to selection rather than to some other force (drift, mutation). Some drift is bound to happen when the lines are founded, because you are selecting a subset of the population, which is actually a good thing. Each line gets a random subset of genes which then evolve together. This potentially allows discovery of different genetic pathways to the same adaptive response.
The background and lots of examples are in Garland and Rose, Experimental Evolution (UC Press, 2009).


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Are Italians genetically separated from other Europeans?
I was reading the Wikipedia article about the genetic history of Italy, and I found it interesting. There are, however, a few things that puzzle me, because they seem to contradict each other.
For example, it states that, because of the Alps forming a natural barrier, Italy is, along with Finland, a genetic island in Europe. Does this mean Italians and Finns are genetically separated from all other Europeans, and to what extent?
Later in the same article it says that

In Italy as elsewhere in Europe the most common haplogroup is haplogroup H

which would seem to contradict the genetic island theory. After all, Italy has been colonized by countless populations throughout its history, which might suggest the Alps didn't really stop the gene flow from neighboring countries (not to mention that other European countries are equally separated by mountains, like Spain).
I have no background in genetics whatsoever, so please excuse my ignorance. I am just trying to understand an article that seems very confusing and throws lots of different information together.

EDIT: Thanks for the quick reply! I've found another link, which seems to be more reliable than Wikipedia's unverified claim, though it still sounds like a weird theory to me.

Please don't ask many questions in a single post. Split them up into separate questions instead. I have removed the second question, please post it again separately.

you have to remember that in most human history people just didn't move around like can now.  population haplogroups do migrate, but slowly enough to identify their movement.  The Alps didn't stop migration, but they would have kept the flow down significantly compared to the overall migration rate in the french areas, connected by rivers and flatter terrain.

This makes me think of a report of a healthy village in Italy where 400 people were 90% descended from a Northern European couple and were genetically studied because cancers were very rare in the village... Also note that walking from Genoa to Marseille has a maximum elevation of 550m. Alpine populations can be fairly insular, but Italy is very diverse.

No, Italy is not a Genetic island but Sardinia is;

There is no such thing as a genetic Italy or an Italian (genetically); Italian groups (North Italians / Central Italians / South Italians / Sicilians / Sardinians) are diverse and do not cluster with each other (DiGaetano et al 2012 / Nelis et al 2009) in PCA-plots; In fact Italians are just as little related with each other (IBD sharing) as they are to other nations (Ralph & Coop 2013);
In general North and Central Italians cluster closest to each other and to Greeks and Albanians while South Italians cluster between Greeks and Cypriots (Lazaridis et al 2013);
What is truly a 'genetic island' is the island of Sardinia; The Sardinians are an isolated population and only cluster with old Neolithic-Europeans (Keller et al 2012 / Lazaridis et al 2014 / Gamba et al 2014 / Haak et al 2015), reason being they are ~90% genetically identical (inheritance) with the old Neolithic-Europeans (Haak et al 2015 / Sikora et al 2014);
As for the quoted study i.e. Lao et al 2008; It is not clear (to me) what the sample make-up of IT1 is; In Figure S2 one can clearly see its samples clustering all over the place; Some samples cluster in between Greeks (EL) and Swiss (CH) where as others are completely isolated; I assume that these samples are from all over Italy incl. Sardinia and that the isolated samples are the Sardinian ones; Lao et al is a study from 2008, and probably because of such results, more recent studies tend to label the Italian samples according to region (North Italians / Tuscans / South Italians / Sicilians / Sardinians) in order to have a clearer insight;

So the real 'lesson' is that Italians, despite being politically ONE nation, are in fact however genetically diverse i.e. not one people; And Sardinians are an isolated old-stock, in fact the last Neolithic remnants in Europe;
Ultimate advice: Dont bother with Wikipedia and whatever someone has written there; Read the studies themselves (first hand), thats the most reliable;
Some inside into Italian genetics:
K=7 and K=9 Admixture analysis (incl. North Italians / Sardinians)
Di Cristofaro et al 2013
http://s3-eu-west-1.amazonaws.com/previews.figshare.com/1251712/860/p_01.png
K=10 Admixture analysis (incl. Tuscans / Sardinians)
Behar et al 2010
http://s14.postimg.org/g7agga7e9/u1u11.png

That's an interesting answer. Can you add links to the cited papers?

I'm afraid that the "genetic island" idea is almost certainly wrong because

The wikipedia page offers no evidence for this claim:

Following scientific research carried out by Dutch geneticists, Italy has proven to be one of the last two remaining genetic islands across Europe (along with Finland), this due to the presence of the Alpine mountain chain that, over the centuries, has prevented large migration flows aimed at colonizing the Italian lands.[citation needed]

Remember that any random idiot person can edit a wikipedia page. If there is no supporting reference for a claim you find in wikipedia, you should treat it as suspect.
As you point out, the Alps only block entry on the north of Italy. Italy, however, is a pretty large peninsula, completely open to the sea along its entire length. For example, here is a map of Greek and Phoenician colonies circa 530bc (source):

As you can see, significant parts of the coast of what today is Italy were colonized by Greeks and Phoenicians. There were many subsequent waves of migration and/or invasion by various tribes such as

The Lombards (image source):

The Visigoths who sacked Rome in AD410
The Ostrogoths whose kingdom in its prime contained most of modern Italy

The many Jewish communities in Italy that go as far back as the Roman era.

An article by Brisighelli et al. (which is actually quoted at the very top of the Wikipedia page you linked to) mentions that (emphasis mine):

A total of 583 individuals were sampled from across the Italian Peninsula, from ten distant (if homogeneous by language) ethnic communities — and from two linguistic isolates (Ladins, Grecani Salentini). All samples were
[...]
Italy shows patterns of molecular variation mirroring other European countries, although some heterogeneity exists based on different analysis and molecular markers. From North to South, Italy shows clinal patterns that were most likely modulated during Neolithic times.
[...]
Before the Roman conquest, ancient Italy was characterized only by the presence of Indo-European populations living in the Italian Peninsula since the second millennium BC, corresponding to the period between the Iron Age and Romanization. During all this period there were also increasing contacts with the Phoenician and Greek colonists: the former being largely present on the coasts of Sardinia and western Sicily and the latter in Southern Italy.
[...]
At the beginning of the first millennium BC the following native tribes could be distinguished on the Italian territory: the Ligures, on the coast that bears their name, in the northern Apennine valleys, part of the pre-alpine valleys and the western Po Valley; the Sicani, in the interior of Sicily; and the Itali, in present-day Calabria (from whom comes the name ‘Italy’, which was to be extended to all the territory of the peninsula). Besides the already mentioned Terramare tribe, on the southern edge of the Po Valley, and the Villanovans, probably from Eastern Europe who settled throughout Central Italy, there were also the Umbrians to the east of the upper basin of the Tiber. The Veneti, who occupied the territory that still bears their name, originally came from Illyria as did the Messapii (now modern Salento or South Apulia) and Iapyges, who settled in present-day Puglia (Apulia) 5. Many other populations of Central-Southern Italy were created by the mixing of local and foreign elements dating back to the previous millennium; it is the case of the Sabines and Latini who settled in Lazio together with Falisci, Aequi, Volsci, Hernici and Ausones. The interior of Abruzzo was dominated by the Vestini, Paeligni and Marsi, while the central Adriatic coast was populated by Picentes, Marrucini and Frentani. The Apennine area of Molise and Basilicata was peopled by the Samnites and Lucanians. In Calabria and Sicily there were also the Bruttii and Siculi.
[...]
Molecular indices indicated that most of the Italian samples show diversity values that are comparable to other European populations. However, some differences were shown to exist, especially in isolated Ladin populations.

References

Brisighelli F, Álvarez-Iglesias V, Fondevila M, Blanco-Verea A, Carracedo Á, et al. (2012) Uniparental Markers of Contemporary Italian Population Reveals Details on Its Pre-Roman Heritage. PLoS ONE 7(12): e50794. doi:10.1371/journal.pone.0050794

To your point 2 I would also like to add that the fixation with land routes is a recent phenomenon. In ancient times, travel by sea was much easier than travel by land (roads were often few and poor), which meant that distant places linked by sea or rivers could be much more connected (both travel and gene flow-wise) than areas that were geographically closer together but linked by land.

Italians do have much less IBD sharing with other European populations and are more easy to distinguish based on aDNA from other European population, so this enforces the model of the ''genetic island''
''The map also identifies the existence of two genetic barriers within Europe. One is between the Finns (light blue, upper right) and other Europeans. It arose because the Finnish population was at one time very small and then expanded, bearing the atypical genetics of its few founders.
The other is between Italians (yellow, bottom center) and the rest. This may reflect the role of the Alps in impeding free flow of people between Italy and the rest of Europe.
Data for the map were generated by gene chips programmed to test and analyze 500,000 sites of common variation on the human genome, although only the 300,000 most reliable sites were used for the map. Dr. Kayser's team tested almost 2,500 people and analyzed the data by correlating the genetic variations in all the subjects. The genetic map is based on the two strongest of these sets of correlations.
The gene chips require large amounts of DNA, more than is available in most forensic samples. Dr. Kayser hopes to identify the sites on the human genome which are most diagnostic for European origin. These sites, if reasonably few in number, could be tested for in hair and blood samples, Dr. Kayser said.
Genomic sites that carry the strongest signal of variation among populations may be those influenced by evolutionary change, Dr. Kayser said. Of the 100 strongest sites, 17 are found in the region of the genome that confers lactose tolerance, an adaptation that arose among a cattle herding culture in northern Europe some 5,000 years ago. Most people switch off the lactose digesting gene after weaning, but the cattle herders evidently gained a great survival advantage by keeping the gene switched on through adulthood.''
http://nytimes.com/2008/08/13/science/13visual.html

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Are there cannibalistic k-selectors?
My understanding of r- and k-selectors does not go deeper than the wikipedia article. I understand k-selectors to be creatures that put tendentially more effort and energy into rearing their offspring, but produce tendentially less offspring.
With cannibalism I don't mean eating their own on rare occasions, like after a plane crash in the Andes, but exibiting cannibalism outside of extreme situations.
My gut feeling tells me that expanding much energy into raising an offspring and then eating her does not make much sense, but the world is big, there's lots of different species of animals and most of them are weird once look closely enough.
A good answer explains why one species is a rather to be seen as a k-selector, and in what contexts the cannibalism happens. Since we are talking about a sliding scale of behaviours, the question might as well be read as: What cannibal is the strongest k-selector?

pleas add tags, I don't know what this field of biology is called.

Cannibalism can also help a k-selector. If the female spends a lot of time caring for her offspring and is not eager to have another, why not kill somebody else's offspring to have the mother bear yours. Bears do this. Lions as well.

Interesting question. I am not sure I understand it though. Is it "What are all the possible reasons for killing babies of its own species in a k-species?" or "What are all the possible reasons for eating individuals of its own species in a k-species?" Or is it "Why does a species that practice cannibalism might evolve to be a k-species?", or  "Is it more likely to observe canibalism in r-species rather than k-species?"

"What are all the possible reasons for eating individuals of its own species in a k-species?" is closest.

@mart So it doesn't have to be specific to eating its own offspring? Or does it?

no, not specific to own offspring

My answer is messy and incomplete! I very welcome editing!
As soon as an individual is killed, on might as well eat the dead, but the act of killing might not be for the purpose of eating. We might even say that eating a dead might not only be for the purpose of getting energy and matter but only to clean the living place. And killing might appear in many circumstances. The best way to consider such issue is certainly by using the quite fundamental model of kin selection theory. It fits as much to species that gain energy by eating the dead or not. The difference imply just a minor change in the payoff matrix.
The decision of one (the actor) to kill (and eat) another individual (the recipient) depends on:

the relatedness of the actor to the recipient (note a relatedness is not necessarily symmetric)
The competition this recipient causes on the actor
The cost (energy cost, risk of being injured or killed) of killing

Many species (ants, wolves) might kill or injured individuals of the same species, when for example there is competition for reaching a status in the hierarchy.
I think that the definition of k-selected species is not very accurate. First because there are limit cases. But also because r and k can relate to the function that describes the population growth of a species or might describe the amount of energy the parents spend into one offspring which is not necessarily the same thing. If we think of the parental care, we can think of the lions. When a male win the right to access to the females of another male, he will kill the babies that might be potential competitors to his progeny (because they take energy from the females or because females are not fertile before having lost their babies).
Killing its own offsprings is certainly not less common in k-selected species. You might think it is such a big cost to kill one of its own offspring when one has few offspring but what is important is to think in percentage. Killing 50% of the progeny has the same cost of an r-selected species than for a k-selected species.
If one has a probability of 10% to be able to raise its offspring to adult age. But this probability raised to more than 20% if it accepts to kill an offspring to save energy for the next offspring, then it wins by doing so. THen we might ask: "so would one make an offspring if it intends to kill it?"
Well it does not necessarily intend to kill it. It might need to decide late in the season whether or not it has better to kill its own offspring.
Then, maybe offspring might be used as a reserve of energy and matter for its siblings or for its parents.


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What is the most recent non-primate ancestors of humans?
If we go down the evolutionary tree of humans, (Homo sapiens, Homo erectus, ...), what will be the first non-primate ancestors?

Note that humans did not evolve from the tree shrews, both they and we share a common ancestor. See here for more.

I see that you edited the question to include "ancestor". @terdon makes a very good point. We do not think of ancestors in the fossil record in the same way that a person has ancestors (parents, grandparents, etc.). The most recent common ancestor of primates and tree shrews is neither a primate nor a tree shrew. There is no name for that taxon.

According to the Tree of Life, tree shrews (Scandentia) are the sister group to Primates. So Scandentia are the most closely related extant mammals that are not Primates. Here's an image:


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Regulation of the replication of mtDNA at embryonic level
While reading an article on mitochondrial inheritance I came across this link.
The results state that mitochondrial DNA replication is regulated in different cells of an embryo at different levels. How is this regulated?

I'm sorry for not having mentioned the key word GENOMIC earlier. That made the question unclear, as I understand.

As for the synchrony of mitochondrial and cellular division it would be of a great help to explain.

This is a topic which seems to be not very clear yet. References say that this is obviously dependent on the tissue and the "gene dosage" seems to play a role, and that this is probably regulated by a yet unknown factor (see "Mitochondrial DNA replication: what we know." and "Replication and transcription of mammalian mitochondrial DNA.").
Newer articles suggest that it depends also on the transcription of the mtDNA (see "The interface of transcription and DNA replication in the mitochondria." and "Mitochondrial transcription factor A regulates mitochondrial transcription initiation, DNA packaging, and genome copy number."). I will see, if there are other interesting publications, if there are problems with accessing these articles, let me know.

Mitochondria divide by binary fission. The regulation of this division differs between eukaryotes. In many single-celled eukaryotes, their growth and division is linked to the cell cycle.

I guess the concern of the asker is to understand the type of regulation and not whether it is regulated. Can you please elaborate on the mechanism of this synchrony between mitochondrial division and cell cycle.


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Problem with bacterial transformation with electroporation
I have a problem with a bacterial transformation of a yeast gene that I can not solve.
I isolated yeast DNA and did a PCR to get my product. I am using pCGCUm vector with a GFP construct. I digest both my plasmid and my product with SmaI and ClaI for 4h each. After that I purify the product from a gel and perform a ligation with high efficiency T4 ligase for 30 minutes at 22°C. Then I transform my electrocompetent cells with electroporation (2000V, 5ms), recover it in 0.5ml LB for 2h and plate it on LB-A plates.
First time I only did:

insert1
insert2
Empty vector

and it did not work (empty plates for insert and confluent growth (after 18h) of some weird colonies on EV).
I did the same ligation again and this time I included controls
This is what I did:

insert1
insert2
Empty vector
negative control (EK cells + electroporation)
positive control (EK cells + undigested vector)

Now I got:

insert1 (no colonies)
insert2 (big colonies a lot of them)
Empty vector (big colonies a lot of them)
negative control (EK cells + electroporation) (no colonies)
positive control (EK cells + undigested vector) (big colonies a lot
of them)

The negative control suggests that my competent cells are ok, at least they are not resistant to AMP.
What confuses me is that even with positive control I get some weird big colonies - if I got nice transformants here I would assume that either digestion of ligation was not successful, but now I doubt that).
Another thing that is weird is that now I got that colonies also on my insert2 plate.
The scan image of big colonies looks like that:

I have a question if anyone has had that kind of colonies? I was thinking of maybe having contaminated cuvettes for electroporation but I had them in 100% EtOH for 3 days and then dried in fume hood for 3 hours before electroporation.

Some questions: 1) Can you describe the pCGCUm plasmid in more detail? antibiotics, presence of ccdB gene or MCS, etc. Can't find it on the web. 2) What are EK cells? 3) Where did you get your transformation protocol? From someone else in your lab who's had it work for them? I've been told not to let cells recover from electroporation for more than 1 hr because they may lose the plasmid during prolonged recovery, but if this has worked for a labmate then that's not an issue. 4) Have you repeated the 2nd expt (with the extra controls)? If it's contamination it might not reappear a second time.

I agree those big colonies do not look normal. It's possible that your competent cells are contaminated with some strange strain. You could try plating serial dilutions of your competent cells (at least 10^6 fold dilution) until you can see individual colonies, and see if it's a mix of normal looking and weird colonies. Again, ask other people in the lab if they've had contamination issues--one time I struggled for a long time with low tranformation yields until I learned someone else was having the same problem, and we made a new batch of competent cells and were fine.

Hey. Thanks for comments. pCGCum is 5.5kbp long. It has an ampicillin resistance and MCS long 53 nucleotides - SmaI on 8 and ClaI on 47. EK cells are electro competent cells(sorry I translated it from german). The protocol is standard protocol that has been used in our lab for long time now and has worked ok till problems appeared lately. At the moment it is only 2 people that were actually doing it and other one have the same problems with that colonies.

We made a new batch of electro competent cells and alkaline phosphatase is new. Ligation kit was changed before my experiments. I will make new batch of chemical competent cells and try with those and hope to have some success. I will also do plasmid prep on insert2 plate where I have the colonies and try to digest it, maybe they are just "weird positives" - which would still not give me the answer but at least I will know something more. I will also plate different dilutions next time repeating exp.2 with our EKcells, EKcells from other lab and chemical competent cells.

I did try another transformation:

again with our electrocompetent cells
Cells form other lab
chemically competent cells I made

The good thing is that on chemically competent cells there is no weir colonies, however efficiency of transformation was very low, but still, I think it is ok - will confirm it in next day if I really got the right insert.
On the other hand my electroporation still did not clear out. I got colonies on both our cells and cells from other lab, however in much lower amount there. I will try to change the cuvettes and try to use different electroporator as this seems to be the only thing that could make problems - although I am still not convinced about that :)
Thank you for your help and I will report the progress so if somebody else will have that problem again he could maybe benefit from my findings.

So, the answer is clear now. The contamination was in cuvettes. Some kind of bacteria that can survive 70 and 96% Ethanol, probably in form of spores. What I did is I putted cuvettes in LB medium for several hours so I got growing cells, than soaked them in 70% ethanol overnight and rinsed them with 96% EtOH for 2 hours. Than I added LB to cuvettes, "recovered it on 37 for 2 h and plated on LB plate. Plate was clean next day and also I did the normal transformation which worked ok!

Do you re-use cuvettes? If you do so, you should at least incubate and wash them in 0.1M HCl to get rid of DNA contaminants before washing them with 1% SDS and getting rid of all the detergent afterwards with a lot of water. We stopped doing this and either use the cuvettes only once when we really have to do electroporesis and the rest of the time chemical competent cells work fine.

Yes, we reuse the cuvettes and we only washed them with ethanol. Everybody in the lab were doing it that way, but with all the problems I had with cloning I will use chemically competent cells from now on. The efficiency is the same and I like it more.


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Could alcohol be used as an antibiotic?
I was thinking about how alcohol is used to sterilize, and I was wondering if it would be possible to use alcohol as an antibiotic? if it kills bacteria, couldn't (conceivably) alcohol in the bloodstream kill any bacteria there, and the alcohol would be cleaned out by the liver later? Or would the concentration of alcohol necessary be too high and cause poisoning?
Sorry if this has an extremely obvious answer, but all of the results I found in Google were about drinking alcohol while on antibiotics.
Could, in theory, alcohol (delivered in any way) be used as an antibiotic? If so, how? If not, why?

No.  The CDC recommends using a hand sanitizer at at least 60% ethanol.  Most preparations used in the lab are 70% ethanol.  Humans start to risk death somewhere around the 0.3% alcohol level (BAC), especially around 0.5%.  People have gone higher, but even 1% BAC would be completely useless against bacteria.  You'd be dead long before the bacteria died.  Besides, if you've got bacteria in your blood, you've got bigger problems than a simple infection.
In a pinch, though, it can be used as a topical antiseptic, as per this fun article.

Thanks. I didn't think that this would work, but I thought I'd ask some people who actually know.

Out of curiosity, what makes alcohol poisonous to humans? Is there any chemical that can inhibit that?

@Linuxios that deserves a question (well, two actually), it's not really something that can be answered in 600 characters.

Minor quibble: as elaborated in the rest of your answer, the answer is arguably "technically yes, but it would kill you".


text:
What makes alcohol harmful to mammals?
During the alcohol metabolic pathway, harmful by-products are made like acetaldehyde, hydroxyethyl, superoxide anions, and hydroxyl radicals. How do these toxic compounds harm our tissues? Many of these intermediates are the compounds that are responsible for hangovers, but are they also the molecular cause of liver damage?

This answer gives a nice explanation of hangovers.

Thanks! I've tweaked the question so that it's not a duplicate.

It wasn't a dupe before either, just figured you might be interested.

That particular sentence was a bit 'dupy' when I read it back. The other answer was interesting though! I'll stick to clear liquors if I don't want to feel so bad in the morning! It seems we are still unclear of the actual cellular damage. The article mostly discusses the dehydrating factors. Hopefully someone will be able to post newer answer with more molecular information.

Superoxide radicals , basically damage the lipid bilayers. affecting the efficient gradient.
for example H+ ion gradient across lipid bilayer in ETC is very important. this gradient is affected if superoxide radicals are present.


text:
Natural enemies boost deep evolutionary fitness?

"Two species that reduce one another's fitness on microevolutionary
  (short-term) timescales can increase each other's macroevolutionary
  (long-term) fitness"

Opinions?
Intuitive Examples:

Body Size - Predator-prey relationships or interference relationships
(fighting, e.g. for territory) are often documented to put seletion
pressures to grow in body size, often a positive feedback loop. In
the short term the presence of the natural enemy in this case often
leads to decreased population fitness (probability of being
eaten/probability of fatal conflict over territory). But in the long
term such large body sizes acts as an advantage over potential future
enemies of the same kind. This is particularly relevant to marine
ecosystems - where the size of fish often determine where in the food
chain that fish is.
Speed/agility - Predators and prey that rely on speed/agility to
capture (predator) or escape (prey) undergo an evolutionary arms race
leading to positive feedback of speed/agility boosting morphologies.
In the short-term the natural enemy of the prey acts as a burden to
population fitness (probability of being eaten) but in deep time the
prey has acquired morphological advantages over potential future
natural enemies such as competitors for resources.
Venomousness -Why do venomous animals live in warm climates?

The question is interesting.
I am afraid about the use of the word "fitness" in you question. The fitness is usually defined as the number of offsprings an individual can sire in its lifetime. Most often we talk about relative fitness which is the relative number of offspring in an individual can sire in its lifetime compare to the individual in the population that sire the greatest number of offsprings.
How would you compare fitness of individuals between different species? Just by counting the number of offspring they left behind? Or did you mean that most fit species are actually those species that can outcompete others? If yes, then I would say that saying

Two species that reduce one another's fitness on microevolutionary (short-term) timescales can increase each other's macroevolutionary (long-term) fitness

can be restated

Two species that reduce one another's fitness on microevolutionary (short-term) timescales can increase each other's macroevolutionary (long-term) species competition ability

and it is actually just a special case of

One species that evolve under many strong selection pressure ends up being a very competing species.

The explanation is the following:
One species that undergoes one selective pressure, will see its genetic variance decreases. The weak individuals will disappear letting places to the strong individuals. Then newly arisen variance (mutation) will come up only in the strong individuals (as the other ones disapeared). This is what J.Gould meant by saying "Natural selection creates the fit". As a consequence a species that undergo many successive selection pressure will end up with purified and very strong genotypes while, in a species that were free to evolve without selection they would have evolved many different genomes with some good mutations nested in very poor genetic background.
You could as well use the concept of lineage selection and states that lineage that succeeds to outcompete others will thrive while species that suffer from competition with other species will get extinct. As a result, if you compare all species that were in competition with another species in the past with all species that were not in competition, you will find out that those that were in competition are more resistant to competition. But I guess this is not exactly what you meant when saying: "Two species that reduce one another's fitness on microevolutionary (short-term) timescales can increase each other's macroevolutionary (long-term) fitness"
Finally you might think of the ability of species to evolve better. This ability is called "evolvability" (see this book for example). You might say that species that undergo many selection pressures were selected to resist to newly arisen selection pressure. As a result, species that are under competition might end up with a greater evolvability than those that were not under selection. Resulting that species that were under competition will tend to increase to always increase their fitness in the long term.

I believe I was not just addressing "competition". E.g. Having larger body size does not simply increase competitive ability, and may sometimes reduce it (increased resource requirements). The ramifications of having certain positive feedback loops in morphology change has a more general effect. Indeed fitness was a word I chose since I could not choose another - perhaps I mean "fitness relative to future potential phenotypes". Fitness is not just restricted to #offpspring, that is only the "life histories" definition isn't it? http://en.wikipedia.org/wiki/Fitness_(biology)

The wikipedia link you provide says: "Fitness is a central idea in evolutionary theory. It can be defined either with respect to a genotype or to a phenotype in a given environment. In either case, it describes the ability to both survive and reproduce, and is equal to the average contribution to the gene pool of the next generation that is made by an average individual of the specified genotype or phenotype." and this wiki article does not use the word life-history.

Fitness is usually considered to be the (relative or not) number of offsprings one can produce in its lifetime (or per year). It is also often considered to be number of fertile offsprings or even the number of grand-children. This concept even if it is very central. Here is a discussion concerning the concept of fitness but the discussion is still open I think.

But anyway. What is the definition of the words you're using in "Two species that reduce one another's fitness on microevolutionary (short-term) timescales can increase each other's macroevolutionary (long-term) fitness"?

Ah thank you @Remi.b. To make the "life histories" clearer, I should have asked: "Arn't you talking about reproductive success?" (the life histories definition of fitness): http://en.wikipedia.org/wiki/Reproductive_success. As opposed to the population geneticists definition (my area of research) that considers relative changes in allele frequency.

Microevolution and Macroevolution are two fields of evolutionary study. One deals with short-term dynamics such as changes in allele frequency, the latter deals with processes that span multiple speciation events.

Yes, I agree with you definitions of micro- and macro-evolution. I don't think there are any different between fitness and reproductive success except the one that a particular person wants to make. Sometimes reproductive success is considered non-relative while fitness is relative. I still don't really understand what you call "life history definition of fitness"

let us continue this discussion in chat

authors: เน็ตฟรี buy อาทิตย์


text:
Biodiversity is restricted by genome combinatorics?
Me and some friends are interested in opinions for the following:
Conjecture

The maximum number of species must be limited by the maximum
  combinatorial/permutational space that can be occupied by DNA. Thus if
  there is a maximum physical genome size this is what will determine
  the maximum number of species that can possibly exist.

Explanation
E.G. say maximum number of DNA base pairs able to fit in a genome was $3$, each base pair can be one of either ${A,G,T,C}$. Then there are $4^3 = 64$ possible combinations of genomes. Extrapolate to genome sizes of $x$ base pairs, then there are $4^x$ combinations.
Questions
Would it be possible to claim that the underlying "blueprint" that codes for living diversity sets the absolute maximum for the total "diversity space"?
**Does it make sense to define the total number of species life can achieve with the simple function:
$S < 4^x$, where X is the maximum genome size measured in DNA base pairs?**
Notable Comments

@Shigeta: for $S<4^x$ the combinations involved quickly dwarf the
  number of atoms in the universe at ~10^80.
@rg255: Even at this simplification of: $S<20^{x/3}$ there are
  $1.024e+13$ possible combinations with just 10 codons, many many more
  than there is likely species in the world.

You would have to alter the current library of amino acids- the 64 nucleotide patterns you mention currently only code for 20 amino acids

Also this is not a question

Please try http://skeptics.stackexchange.com for this kind of discussion. On this page, opinion based questions are not allowed.

@skymninge Such a question would be off-topic on Skeptics, the site requires notable claims for any question.

Issues I have with this idea: 64 nucleotide combinations does not make 64 different results (just 20 amino acids), you assume one nucleotide difference will make a large enough reproductive barrier to create a species (if that were the case almost every individual organism alive could be called a different species), the length of DNA is theoretically infinite so there is no upper bound, environmental effects and gene*environment interactions can bring about differences in phenotype which could make "species" from the same DNA sequence, and not all combinations of DNA will be useful/viable

I don't think hello_there_andy is suggesting something he had thought about and ask for our opinion on his theory. There are several criticism to draw on his theory but this is the whole point of his question. This type of calculations is common in biology and he does not know about it. It is worth answering this question by saying what are the problems with his concept of species and what are the problems with the calculations of DNA capacity to hold information.

This site is not for opinion based answers, the question made little sense - no question was actually asked - and, there are fundamental flaws in the biology of the post

Well, there's a tag theoretical-biology on Biology.SE. This question has to do with theoretical biology. And as @MadScientist said it would be off-topic on skeptics. But anyway. The question is already fully answered!

It is a basic maths exercise put in the background of simply reducing species down to nucleotide differences and ignoring various other biological concepts and principles. I agree that "If the genome is limited to x bases then there is only 4^ x combinations of bases."

Hello, Thank you all. Sorry about not making it sound more like a question, and thank you for editing it to fix that. My honest intention was to learn from criticisms. I have learnt an incredible lot thanks to answers. It is not just a mathematical excercise. I realise the 4^x is a gross over estimation... but I am sure you agree "there cannot be >#species than there are combinations of genomes"

I must say this is my second question so far, and I will be more disciplined now. I did not acknowledge the level of freedom I have till now, please forgive me!

The edits made are good, but the robotics bit is a little out of place, so if that is better expressed as a biological question I will vote to reopen. Just as a note, given that a codon of three bases codes for 1 of 20 amino acids the better simplification would be 20^(x/3) ... at just 6 bases (2 codons) the number of possibilities is 10 times lower (400 vs 4096). Even at this simplification there is  1.024e+13 possible combinations with just 10 codons, many many more than there is species in the world.

Thank you @GriffinEvo I must argue that non-coding DNA has many functions: http://www.genome.gov/10005107

That's a very good point, the possibility the non-coding dna might act on a base-by-base level rather than in triplets is something I hadn't considered

Ah yes, I suppose in all honesty I felt an opportunity to add that diagram since It was something I made for a presentation. I have removed the diagram, hope that would solve the issue!

I have now voted to reopen because I think this is now a question set within a biological framework.

let us continue this discussion in chat

@GriffinEvo Thank you, I have already learned much from the discussion too, cheers. Also I have further edited the question with better organisation and headings.

Just wondering...Can there be a new species without a change in coding sequence and only change in regulatory sequences ?

@biogirl yes provided that change brings about a reproductive barrier - up and down regulation of a gene can produce variation in phenotypes

@GriffinEvo WOuld that have any implications on this question ?

It has to be said, the definition of what a species is, or in another way the question: "what is a species" is actually highly controversial... mainly there is a divide among "Niche species" and "Reproductive species" - in the former the critical principle to read about is "Competitive Exclusion Principle" http://bit.ly/cqEkle the one @GriffinEvo hinted is the latter.. which is a more practical definition that is if two organisms fail to make viable offspring they are seperate species - indeed there are at least 20 species definitions e.g. http://en.wikipedia.org/wiki/Species

@biogirl My argument here simply puts an UPPER LIMIT to the whole debate. I have no idea how many species there can be but I can say with some confidence that there cannot be more than 4^X species. That is.. the #species that can ever exist is <4^X

I asked the question in a similar way in the Stack Mathematics forum: http://bit.ly/1bSNwNu

@GriffinEvo There are 22 proteinogenic amino acids (including selenocysteine and pyrrolysine), not 20. Just playing the pedant here but I did my thesis on predicting selenocystein-containing genes so it's kind of a personal issue for me :).

@hello_there_andy you should read Chp 3 of The Blind Watchmaker. I feel it is relevant to your question.

Cheers Biogirl and GriffonEvo, since joining Bio exchange I have collected a big "to read" list :D

Yes, we can say the number of species is limited as you conjecture.  However, quick estimation shows that the limitation has no apparent usefulness:
A reasonable estimate of the largest known genome is 150 GB (150,000,000,000 or 1.5e11 nucleobases).  The limit would be 4 raised to that power.  That limit is so high that it is too large for most calculators to calculate.  For instance. Wolframalpha.com fails to calculate 4 raised to 1e11, its maximum being about 4 raised to 1e9.  The result of 4 to the 1.0e9 power is about 1.0e602059991 or 10 raised to the 602059991'th power.
That rough result, 1.0e602059991, is so enormous that it is exponentially greater than the number of atoms in the universe (which is less than 1.0e100).  Hence, assuming the definition of species requires an organism to use at least one atom for its body, there is no consequence to saying the number of species must be less than this number.
The number of possible combinations of nucleotides is so outrageously large that it does not constrain the number of unique individual organisms or species.

That's an interesting conjecture about the total amount of genetic variation that is possible. I would modify a few things. First, since the size of genomes varies greatly among organisms (from 0.5 Mb to 15 Mb just for prokaryotes), there should be a fifth character in your set, representing the absence of a nucleotide.
There are also issues of whether various combinations are equivalent. For instance, bacterial genomes are often circular, so if we could convert one genome into another simply by rotating it, we would consider those genomes identical. For example, if the genomes each had 99 As and 1 T, it would not be meaningful to say that they are different just because of the location of the T. I think this would require use of the multinomial coefficient to count the number of identical variants.
Regarding your main thesis, your use of the term "species" has no relation to how that term is used among biologists. Biological species include genetic variation, so each of them would include many of your species. Also, one criteria for identifying species is the clustering of these sequences (and the absence of other, intermediate sequences). This implies both that many possible DNA sequences simply cannot produce a viable organism and competition among similar genotypes is an important aspect of species identity.

+1 Very good answer. Of course there is the fact that DNA contains more potential information that what a protein can contain. And therefore, the total space possibility of proteins from x base pairs is lower than $x^4$. the OP might be interested in the [quasi-species theory][http://en.wikipedia.org/wiki/Quasispecies_model] presented in [this book][http://www.amazon.com/Evolutionary-Dynamics-Exploring-Equations-Life/dp/0674023382] for example.

The OP might as well be interested to the concept of robustness ([this book][http://www.amazon.com/Robustness-Evolvability-Living-Systems-Complexity/dp/0691134049] for example)

I'm going to define a species according to the biological species concept, probably the most widely "accepted" species concept where a species is a group of individuals that reproduce, or have the potential to do so. Using a simplified example I will show you that gene*environment interactions affecting phenotype can allow separate species to occur despite being genetically identical.
Imagine a very short section of DNA which affects seminal fluid proteins in a fruit fly. It is  just 10 bases long. The first three triplets of bases code for a protein constructed of three amino acids which affects the females behavior upon receipt of the male ejaculate. The final base regulates the expression of the first codon and is sensitive to developmental environmental factors (let us say nutrient richness). When the development of the male fly is in a nutrient rich background the section of DNA produces all three codons in equal amount to construct a protein and that protein stimulates the female to release eggs for fertilization. If we take a genetically identical male reared in a nutrient poor environment then the regulatory base increases the production of the first codon's amino acid. This makes a different protein structure which no longer stimulates the female in to releasing eggs, therefore the males from nutrient poor backgrounds can not reproduce with the females from a nutrient rich background, and these two genetically identical populations are different species.
Thus I would say, no, the theoretical maximum number of species is not capped by the length of the DNA. However, given that just the potential number of bases (current highest estimates recorded are 150,000,000,000) far far far exceeds the number of species in the world, we can say that the number of base combinations is not the limiting factor on the biodiversity we see. That is down to evolution (selective and neutral processes). Phenotypes determine the ability of two individuals to reproduce, phenotypes are the result of more than just genetics:
$phenotype = genotype + environment + genotype*environment$
Further, as @mgkrebbs has already stated, the number of possible species (given by $4^x$ when $x$ is 150,000,000,000)  is, not only far more than the number of species that do exist, but also far more than the number of atoms in the universe. Assuming each species requires at least one atom then the number of atoms available will halt increasing numbers of species before the number of possible base combinations does.

Coupled with @mgkrebbs answer of atoms in the universe, I have been convinced to a great extent how irrelevant 4^x is. Thank you all for giving me the perspective!

the combinatorics involved quickly dwarf the number of atoms in the universe in fact.  ~10^80

Nobody would consider those flies to be different species. Since there are no females that will mate with the "nutrient poor" (NP) males, those males do not form a separate species -- they just fail to reproduce. Even if the NP males could mate with NP females (to the exclusion of other males), the NP population would not be a separate species because it would still maintain the potential to reproduce with the other members of this species. The isolation of the NP subspopulation from the rest of the species would be transient -- similar to physically separating them.

Maybe you could imagine some sort of feedback loop that would stabilize the mating preferences -- perhaps having been born on a certain type of fruit, they preferentially mate and lay eggs on that fruit. However, I still doubt they would be considered separate species if all that was needed was to transfer larvae between fruit in order to change the preference (which would probably happen on occasion in the wild)

@adam.r sorry, it is a gross over-simplification and to keep it simple I made the (unstated) assumptions that NP females would only be responsive to NP males, and that these environments were isolated during development - artificially bringing adults of the two groups together to mate would then lead to the conclusion that they are separate species as NP<->NP and NR<->NR matings would work... bit of a large jump but, like I say, it is an oversimplification. I should have been clearer in the answer but wanted to keep it simple.

@hello_there_andy this answer may be a little related here... http://biology.stackexchange.com/questions/13834/what-are-some-example-of-genes-that-code-for-multiple-proteins

Well... this is true in the same way that books are constricted by letters. We could have more letters and create more combinations with the same length of DNA. But, like books, there are just so many combinations.

Nice that you spotted the similarity with books. In fact it is more general than that.. it goes for the whole of language, music, computer programs and perhaps all kinds of technology (think about the periodic table).

Well... kind of. With all of those things you are true... except for music. I don't mean to be nitpicky; actually, the science behind the repetitive nature of music is quite fascinating. See this link for more info. http://www.youtube.com/watch?v=DAcjV60RnRw

I guess I simplified music to a string of notes and timings. E.g. the possible number of ways you can create a finitely long sheet of music. More generally I guess there are only a finite number of ways of putting various kinds of sound waves together?

Yes; in the video, it says that in a 5 minute time period (exactly 5 minutes, not a second more), there are about 10^67 different songs.


text:
Do genes play a role in the athleticism and shape of a person?
I am an Indian with a small thin stature with some deposition of fat around my belly.I was wondering if genes play a role in fat deposition in the body for Indian, Chinese, European or African people. Fat deposition may differ among all these people around the belly, thighs etc.
These people also differ in their stamina and speed naturally.

For example here in India in the game of cricket there has been no
  fast bowler out of 1.2 billion people who can do fast bowling
  because it requires a lot of strength and stamina.Also on field
  athleticism is also very bad compared to Australian or South African
  players.I assume that may be its because of some cultural or genetic
  problem.

possible duplicate http://biology.stackexchange.com/questions/3165/athletes-nature-vs-nurture

Here is a book you might like to read

All these characteristics you observe (speed, ability to play cricket, belly size, height, etc…) are what what we call in biology a list of phenotypic traits. Basically, the phenotype is a characteristic of an organism that we can eventually measure or describe (physiological, behavioural, morphological, and life history are four common classes).
In a given population the phenotypic variance (the variance in height for example) can be expressed as the sum of the genetic variance (=the genetic variance underlying the phenotypic trait of interest), the environmental variance (=the environmental variance underlying the phenotypic trait of interest) and the variance due to the interactions between genes and environment (the variance due to the interaction that is underlying the phenotypic trait of interest).
Mathematically expressed it gives:
$$V_{phenotype} = V_{genetic}+V_{environment} + V_{gen X env}$$
Therefore, for each trait there is a given part of the variance that is explained by the environment and a given part that is explained by the genes. We might go a bit further and defined the concept of heritability (in the narrow sense, see wikipedia article for more info). The heritability (in the narrow sense) is the ratio of the phenotypic variance to the additive genetic variance. Mathematically expressed it gives:
$$h^2 = \frac{V_{additive}}{V_{phenotype}}$$
Each trait has it's given heritability. The heritability can be defined only for a trait in a population (which defines $V_{genetic}$) and its environment (which defines $V_{environment}$).
For example, the heritability of height in human is around 0.8 (non-peer reviewed source). Which means that 80% of the variance in height in the world-wide population is explained by the genetic variance. See this post for more info about the concept of heritability
In your question, you also used (before your question was re-edited!) the word fitness (also it is not really present in your question). The fitness is a very important concept in biology, it is the ability of an individual to survive and sire offsprings. Our phenotypic traits influence our ability to sire offsprings and therefore our genes influence our fitness. If the whole phenotypic variance is explained by the environmental variance only, then, even the the trait is under selection, there will be no change in allele frequency due to selection. Note: an allele is a gene variant. If eye color is a gene, brown eyes and blue eyes are alleles (to make it easy). This change in allele frequency is called a response $R$ to selection $S$. This link between selection, the response and the narrow sense heritability $h^2$ is given by:
$$R=S\cdot h^2 $$
So to answer your question. Yes, part of the variance in height, belly size, cricket playing ability, etc… is genetically coded. As the fitness depends on phenotypic traits, Yes, the fitness is genetically coded (otherwise there would have no response to selection). Therefore we're not all equal to playing crickets!

DNA testing is a growing trend in athletics since 2016 to design training regimens by analyzing a range of 40 genes that encode for different physical traits (scroll down for a list of those genes here).
The genes predict mitochondrial and muscle mutations which are good for speed and endurance.
We don't know which physical activities Indians are good at because some countries spend 4.5 million for every Olympic gold medal and India doesn't. They are perhaps better than Japan and worse than Jamaica.
Samoans are physically strong, and have 19% denser bones than caucasians. And they have been surviving maritime life in cold and food-deprived conditions for many centuries. India is the most food-abundant region on the planet, together with East Asia. Nepalese are good marathon runners.
There are two types of skeletal muscle fibers. Fast fibers, which use sugars for fuel and do not require oxygen, kick in for tasks that require maximum force and quick action, such as sprinting. Slow fibers, which employ oxygen-using (or aerobic) pathways, power activities that require endurance, such as long-distance running. A protein called alpha-actinin-3 is made mostly by fast fibers and is implicated in their capacity for rapid force generation. (science mag)
It has been speculated that variations in this gene evolved to accommodate the energy expenditure requirements of people in various parts of the world.(wiki)
https://www.thehindu.com/sport/cricket/indian-cricketers-undergoing-dnagenetic-fitness-test/article20321195.ece
https://www.sciencemag.org/news/2018/04/south-asians-are-descended-mix-farmers-herders-and-hunter-gatherers-ancient-dna-reveals
https://vdare.com/articles/indians-aren-t-that-intelligent-on-average
https://www.researchgate.net/figure/Anthropometric-data-for-European-and-South-Asian-men_fig15_49677710

all_licenses: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/


text:
Bacterial 16S rRNA PCR amplification with universal primers
I am doing an experiment to amplify the 16S rRNA gene from bacteria present in gut contents of fish. After extracting DNA, I perform one-step PCR with universal bacterial primers (27F, 1492R) and I see a strong band of expected length (approx 1500bp). My DNA extraction readings range from 50ng/ul in some samples, to 150ng/ul in others, as measured with Nanodrop.
Does this band represent all of the 16S genes present in the sample? Thus, does this band represent 16S genes from all of the bacterial species present in the sample?
Do I need to perform a microbiome profiling PCR to differentiate and identify all of these species?
Thank you.

The band represents all of the 1500 bp sequences able to be amplified by the primers you chose - no more, no less. Amplification efficiency will likely vary between species/sequences, and while there's a decent chance that your conjecture is correct, further validation will be required to support your assumption.

Thank you MattDMo. So therefore, through DGGE or pyrosequencing of smaller hyper-variable regions of this gene, perhaps all of the different species present in a given DNA sample could be elucidated and identified?

James - yes, something along those lines would be necessary. You've amplified enough of each target (hopefully), so now you can move on to identifying and classifying the sequences it contains. Good luck!


text:
What are the universal laws of Nature concerning animals' behaviour?
I'm looking for very old (centuries) treaty or recent (20 & 21th century) research that state what some of the laws of Nature are, particularly concerning wild animals' behaviour. Such as; every animal does this, no animal does that, etc.
Laws that could be then used to simulate or reproduce natural behaviours or check against unnatural ones.
Added: I'm looking for more simple laws, such as "no (wild) animal eats cooked food". Assuming it's true then humans broke that law of Nature. It's the relation to Nature that interests me. I used the word behaviour in the large sense, as in, what are the laws that animals seem to follow (dos  and don'ts) in the world. I suppose that by looking at what the human is the only one to do we can find lots of those laws.

the reference to the particular work you are referring to is more like a research project seems to me. Different times have different opinions on this and without even a century or millenium to tie it to, it does seem impossible. Origin of Species is not a bad example of such a work.

This question is just far too broad and rather vague in many aspects to be answerable here in this format.

It's really simple, but if nobody has gathered such knowledge before then of course it's going to be difficult to answer.

This seems to be rather impossible to answer! Very fundamental laws of nature are rather found in physics, mathematics or philosophy than in biology.
The theory of evolution is generally considered as a field that brings light to the whole science of biology. T. Dobzhanski famously said: "Nothing in Biology Makes Sense Except in the Light of Evolution" So I guess you might be interested to "laws" coming from that field.
I'm afraid you will some more knowledge in animal behavior than what you can acquire from this simple question before starting simulating. You can easily spend 5 years studying animal behavior and still having much things to learn from that field.
I would guess you might be interested in the concept of selfishness and altruism. I don't know your level of knowledge in biology. The following might seem a bit complicated (although it is a very brief and inaccurate summary of the real science that study these fields), if so, I would invite you learn about basic stuff in biology and evolutionary biology.
Selfishness and altruism
In order to have natural selection, you need:

variation
heritability (of the trait that present some variation)
different reproductive success (or success of copying itself) according to the trait which is heritable.

Following, this idea, an allele that behave altruistically (providing benefit (in terms of ability to copy itself) to someone else to its cost (always in the same terms)) will disappear  to the benefit of the selfish allele and therefore altruistic behavior should never be found in nature.
However, we find altruistic behavior (and that was one of the greatest flaw Darwin could think about his theory):

Because of relatedness (Hamilton's law)

An animal would always behave in order to increase its inclusive fitness (=direct fitness + indirect fitness)

Because of genetically correlated behavior
Because of upper level selection
Because behaving in a way will directly or indirectly influence the way others will behave with you

Now, that's a very big field. I can't develop everything in this answer! If you liked the discussion of selfishness and altruism, you might be interested to read the selfish gene or the extended phenotype.
If you want to go a bit deeper into mathematical formulation you might be interested in evolutionary game theory and hamilton's rule.
Here is a quick formulation of Hamilton's rule on this website

UPDATE (in reaction to your comment)
So, you're not looking for a scientific law/theory/evidence/etc... you're looking for a simple observation in the nature that fit all animals. Here are some examples:

There is no wheeled animals (see this).
There is no animal that migrate every year from Earth to Mars
All animals need a source of organic carbon
All animals possess some flagellates cells (I think)
All animals reproduce (not necessarily sexually)

If you want to talk only about behavior, then you might have some trouble to generalize things to all animals because, there are many non-motile animals, many animals that have no brain, many animals that does not even have organs, … As a side note: It is possible that if you accept that a sponge (which is at the base of the Animalia kingdom see this website if you want to learn about what is classified to be an animal) produce some kind of behavior, you will have to accept that an oak tree also have behavior
UPDATE.
Ah you meant animal in opposition to human?! Humans are included in my above list. If you look for differences then between human and all other animals, than you won't many other things than the brain and the behavior indeed. I hope nobody lost a whole life of work listing what human can do and what animals can't!

Human have a complete and articulate language (I don't fully remember what adjective we use to differentiate human to animal language)
Human are conscious that the are conscious that they are! It is quite hard to show that animals are not!
Humans are the only animals that drive a car!
etc…

You're using the word law in a weird way. You might to have a look to
http://en.wikipedia.org/wiki/Scientific_law and http://en.wikipedia.org/wiki/Scientific_law

That's a bit too theoretic. I'm looking for more simple laws, such as "no (wild) animal eats cooked food". Assuming it's true then humans broke that law of Nature. It's the relation to Nature that interests me. I used the word behaviour in the large sense, as in, what are the laws that animals seem to follow in the world.

@user2534 see the update in my answer.

Very funny. Well I guess that by looking at what the human is the only one to do we can find lots of laws. I hoped that someone had already listed them, though.

@user2534 see my updated answer.

You don't seem to grasp my idea and I don't know how to explain better. But thanks for attempting to answer. It's not about what animals can or can't do (couldn't be a law) especially not about absurd things like going to Mars, having wheels, driving cars. I meant Laws with capital L, as in established by God. Best examples I can think of are: animals may not eat cooked food, animals may eat their own kind (cannibalism), animals may not copulate with their close parents, animals may kill babies of their own kind.

I am afraid you can hardly explain the difference between some silly examples (the example with wheeled animals is actually very interesting and not silly at all!) and yours because there are no difference! Many humans have copulated with there close parents (look at a genealogy of european royalty). I think there are/were some tribute tat practice canibalism and there are many cases of human killing their own baby. I am even wondering if we couldn't find some animals that let their food some time under a big sun in order to cook it.

What do you call a Law with capital L? You can't use God's will to define anything in science.

@user2534 animals often copulate with their parents, they also eat cooked food if they come upon an animal killed by lighting. There are NO "laws" in animal behavior, none for animals and none for human animals either.

ha ha! Don't be impolite! You don't define the word "Law"! And your examples were very poorly found because   both human and animals can perform such behavior. Even if I don't quite understand what your "Law" means, I would tend to say that there are no "Law" that explains difference of behavior between animals and humans because there are no fundamental difference between humans and animals as humans are animals. Note: For further purposes I want to say that this website is for scientific discussion which means using scientific metho.


text:
Determining sequence of oligoribonucleotide

Oligoribonucleotide X was treated with phosphatase (for removal of 3'
  and 5' - terminal phosphates), then with RNAase T1, which cleaves all
  phosphodiester bonds located in a 3' position of guanosine in a
  5'-specific manner.
As a result, oligonucleotides L, M and N were generated in equal
  amounts. Each of them was further treated with phosphatase and
  subjected to alkaline hydrolysis. Results are listed in the table
  below.

Then experiment was modified: oligoribonucleotide X after treatment
  with phosphatase was hydrolyzed with RNAaseP, which cleaves all
  phosphodiester bonds in a 3'-position of pyrimidines in a 5' -
  specific manner.

This hydrolysis yielded five products in approximately equimolar
  concentrations: uridine monophosphate, cytidine monophosphate and
  oligonucleotides P, Q and R. After resolution of the mixture and
  alkaline hydrolysis of these oligonucleotides data listed in the table
  below were obtained.

My thinking : G can not be at the 5' end otherwise we would get a single Guanosine also in the first experiment. G can also not be at the second last from 3' end as we have not got any singles. We have got A and C in a duplet and so they must have been at the 3' end.
I also have trouble understanding the significance of "mole/mole of oligoribonucleotide".
I am trying to fit  in other results but am unable to do so.
A GOOD HINT WILL BE APPRECIATED MORE THAN A DIRECT ANSWER.
Thanks

@Satwik In the above comment - do you mean UMP and CMP ?

@SatwikPasani And by doublet u don't mean they are 1 after the other because I don't think that is possible.

@biogirl It's not clear from what you have written so far that you realise the significance of getting a cytidine rather than a CMP in one of the products - it means, for example that you can deduce the sequence of M, which in turn allows you to deduce the sequence of Q.

@biogirl Did you solve this? If yes, I wanted to know the answer they(the source of the question) provide, to make sure I had the correct solution.

@SatwikPasani I haven't tried this question again as I was busy in other things. Will ask for your help if I am still not able to do it. The answer to this question has not been given in the source. Thanks.

@satwik pasani Nope! I am not able to solve it .Please tell me the answer (not the solution as I want to "reverse derive"it !)

@biogirl I had forgotten my original derivation but I quickly re-derived it. Please check if I have made any errors in haste. $5'-UACGCCGAC-3'$

@SatwikPasani Thanks a lot : I will "reverse derive it"

@SatwikPasani ok i have done it. It's right. Thanks a ton !

Here are a few hints which might help you:-
1) You get free UMP and CMP (in contrast to UMP + C or CMP + U) in the second experiment before alkaline hydrolysis.
2) There are two CMP in "N" oligoribonucleotide, and a free cytidine (and not CMP) in "M" and "Q".
3)Use the fact that all oligoribonucleotides were treated with phosphatase before being subjected to alkaline hydrolysis.
(Referring to your comment, I dont know why do you think consecutive cytidine is not possible).
These are a few hints that helped me solve this. If you want further clarification or the answer, tell me in the comments.
Answer for reverse derivation:- $5'-UACGCCGAC-3'$


text:
Do all humans experience flatus regardless of their diet?
Flatus frequency is affected by diet, so I was wondering if flatus production solely depends on the food eaten, meaning that some people never ever release flatus because they don't eat any flatus-producing food. I guess if they say they don't, they're probably in denial...

Have you read the book Gulp by Mary Roach ? Some interesting flatulence research and other researches regarding gut is brilliantly written !

Eventhough food contributes the most to flatulance, it can also be caused by swallowing air when you chew gum or smoke. It can also be caused by health conditions like coeliac disease and gastroenteritis (reference). So it is indeed hard to believe when people claim that they never fart.

Flatus is a normal product of digestion. To some extent flatus production does not depend on the types of food eaten as some of the gas is nitrogen just from air swallowed while eating. Some of the gas produced may be from bacteria in the intestinal tract. Depending on the species they may not be particularly finicky eaters, and may produce gas from any biodegradable food-item.


text:
Why, specifically, does each generation, on average, improve upon the design of the species rather than degrade it?
In every non-life example I can envision, a copy of a copy is always a degraded or less pure version of the original unless some outside influence acts to correct the copy back toward the ideal represented by the original. Photocopies get blurrier with each generation. Casts from a mold are distorted from the original from which the mold was made. In fact, each cast degrades the mold itself. When data is copied on computers or across networks, parity checks verify that no mistakes were made, but even then, every long once in a while, combinations of errors can cause a false positive in a parity check. So given enough time, the copies would degrade.
In eukaryotes, new individual organisms always begin as a zygote, so in all kingdoms, reproduction boils down to the genesis of a single cell. This involves correctly building the DNA as well as all of the other complex architecture of the cell. Why doesn't this cell degrade like every other example I can think of? In fact, cells are capable of such perfect reproduction that the system generally supports the introduction of additional randomness in order to promote the possibility of productive change. I can think of some probable contributing factors that make this work, but I must be missing something. I can't imagine that this model would actually work the way it does - so well in fact that the design actually improves over time. What am I missing or underestimating?
Contributing Factors (I guess):
Perfect Building Blocks: Cellular development follows a pattern at every level, and ultimately operates all the way down to the molecular level. At that level, nearly all building blocks are identical. Life is built of stable atoms, not something like plutonium, and in the rare event that an atom does change, the result is simply a different kind of atom, which still tends toward a stable form in the long term. Because the structures of life are ultimately made of stable components that are plentiful everywhere in the environment, the essential structures being copied are precise and can be copied precisely. Photocopies and casts are not precise to the molecular level, so copying them is more approximate by nature. Digital data propagation, however, is a very similar process. Bits are also theoretically perfect building blocks.
Fitness Correction: When mistakes do degrade the reproduction process, rather than maintaining or randomly improving upon it, there is a correction mechanism that removes the defects from the process. Those defects do not survive to reproduce. This evolutionary process acts to keep the reproductive pattern focused back upon a theoretical ideal which is independent of a specific physical form to be copied. This seems like the most essential element of the explanation, because it is ultimately only through progression that digression can be avoided, but it is also the part that seems the most dubious. Astronomical quantities of defects would have to be produced before developing just one advantageous feature. I would expect living creatures to be 99% defective with only 1% surviving to breed. I would expect 99.9% of zygotes to expire without being born or sprouting from seed. I would expect all sexual organs (ovaries, testes, stamen, etc.), if not the majority of the whole body, to be mostly dead cells, with just a few successes surviving to fertilization. I would expect 99.9% of the genome to be experimental, almost completely unusable liability to the species. Essentially, I would expect premature death to far outweigh successful life everywhere and at all times. And even so, I would still expect evolution to be even slower than it has been.
Mutation Management Mechanisms: I understand that there are mechanisms in reproduction that decrease the likelihood of mutation in more established and stable parts of the genome compared with sections that are more open for discussion - epigenetic structures, HOX genes, etc. Portions of all genomes have been established and functional for hundreds of millions of years, so I gather that there are mechanisms for protecting them (I suspect probably far more than we have yet discovered).
Note: The numbers I present are fuzzy and are based not on calculations but on general impressions I get of the magnitude of the numbers involved and the relative rareness of useful mutations. Is there any place where this kind of calculation has been performed with more realistic approximations of probabilities?

This question is based on a false assumption. Degradation has been observed in many organisms, and it depends on the specific conditions of evolution. Do a web search for "genome degradation" if you want more info.

They don't, there are a lot more failures than successes.

copies are always degraded, crystal lattices and many other self catalysing reactions  would beg to differ. Also "degradation" in your usages equals mutation which we observe quite a lot of.

You have clearly given this a lot of thought. Unfortunately, as @adam.r said, you are laboring under certain misapprehensions. The quick answer is that each generation does not "improve" on the last. That is a common misconception. In a bit more detail:

First of all, your copying metaphor is a bad one. There was no "perfect original", I expand on this theme at length in my answer here but, briefly, all species are constantly changing. They are not moving away from a platonic ideal of the perfect species (or towards it for that matter), they are simply changing in response to the world around them. What's 'good' today is not necessarily 'good' tomorrow.
Your copy machine metaphor does hold for changes from one generation to the next though. Copying DNA is fraught with errors. There is a huge cellular machinery in place whose only job is to catch and correct those errors. Nevertheless, many get through and result in diversity that can then be selected for or against through the process of natural selection. So, the copies do actually degrade. That, in fact, is the very basis of how evolution works.
Another important point is that most changes are neutral. They have absolutely no effect one way or the other. There are many reasons for this but the main ones are

The vast majority of DNA does not actually code for protein. What it does do is an area of active research but minor changes in sequences that don't code for protein are extremely unlikely to cause a change in phenotype.
Almost all of the information necessary to produce a viable organism is in the genes, and genes represent a very small (~5% in human for example) percentage of the genome. Changes that affect the fitness of an individual are almost invariably found in the coding sequences of genes. This means that of the ~30 billion possible sites for mutation in any given cell, 95% of them (even less actually since only exons count and they're ~2%) will not cause a phenotypic effect.
The genetic code is redundant. Basically, DNA is "read" in "words" of three "letters", the codons. Since there are 4 bases in the genome (A,C,T and G) this means there are 64 possible codons. Each codon specifies a particular amino acid (the building blocks of proteins) and a given sequence of codons will result in a specific sequence of amino acids. However, there are only 22 amino acids, many of which are specified by the same codon:

As you can see in the image above, in most cases, changing the third letter of the codon does not affect the amino acid that will be specified. This means that even for those mutations (changes) that occur in the coding region of genes, the chances are relatively high that they won't actually result in any phenotypic change. If you change the genetic code but the changed codon still codes for the same amino acid there will be no change in phenotype.

As for your contributing factors:

Perfect Building Blocks: Nope, sorry this one is wrong. First of all there is no such thing as a minor change that involves changing an atom. Any change that happens at the atomic level is huge by definition. That kind of thing happens a the hearts of stars and in nuclear reactors. The chemical reactions in our body involve changing molecules not atoms.
There is no such thing as a minor change really, if you replace one atom in a molecule by another, you are significantly changing the properties of that molecule (this is less true for large, complex macromolecules where some changes can indeed be minor). If you were to change, for example, a single atom in normal table salt ($NaCl$) from sodium to hydrogen, you would get $HCl$, hydrochloric acid and not something you want to put in your soup.
There are no perfect building blocks, in biology nothing is perfect, that only happens in math.
Also, the organism does not follow the same pattern from the organismal to the cellular to the molecular (never mind atomic) level. In fact, there are very different organizational principles at play at the different levels and the way that cells are organized (see here for example) has nothing to do with the way that a cell's contents are organized.
Stability is overrated. In fact, our bodies contain loads of unstable (reactive) chemicals, oxygen being chief among them. By definition, chemical reactions involve changing molecules (not atoms, but we don't deal with that level, biological effects tend to be at the molecular, not atomic level). All reactions that go on in the factory that is your body involve the changing of one molecule into another.
Fitness Correction: Actually, at the cellular level, the corrections try to faithfully reproduce the template they are copying from. When a cell copies itself, it will also copy its DNA. It does so by using its own DNA as a template. There is no "theoretical ideal", the cell has no information about the genome of its parent, only its own. As I mentioned above, there are various corrective mechanisms whose job it is to spot errors and correct them. They have no way of knowing whether a given change will be beneficial or harmful to the individual, as far as these processes are concerned, any change is bad and should be corrected. The only thing they do is try to make a daughter cell's genome identical to the parent cell's.
When a change makes it past the cellular level, then it can be selected for or against based on whether it makes the individual carrying it more or less likely to reproduce. This, however, is not a directed process. It just happens. If a mutation makes a male blue whale stronger, it is more likely to be the one that catches up with the racing female and so more likely to reproduce. There is no direction other than the selective process itself. There is no one around comparing new individuals to an ideal and selecting accordingly. If you're better at reproducing than your peers, your genes will be selected.
Actually, many many gametes are discarded. Many cells die. You just don't know about it because they die before you can see them. So deleterious (very bad) changes do occur.
"I would expect 99.9% of the genome to be experimental, almost completely unusable liability to the species." In a way this is true. Despite recent findings, 98% of the human genome does not directly affect the phenotype. It is only the 2% that represents the protein coding parts of genes that has a direct effect on fitness. In fact, in the human genome specifically, there is a short sequence that does not code for any protein and does not (directly, though there are various theories about this) affect our phenotype, that has been making copies of itself and propagating in our genome for generations. Today, this sequence (Alu) represents ~10% of the human genome, that's twice as much as all our genes together!
Mutation Management Mechanisms: The basic protection mechanism you mention in your question is quite simply death. Mutations that render housekeeping genes (like the HOX cluster) inactive kill the organism that carries them. That does not mean they don't occur, it simply means that when they occur, we don't see them because the individual carrying the mutation is dead (see point 3).

Almost ten years later, I just realised that a core part of your answer — “most changes are neutral” — is pretty shaky. What you’re describing there is Kimura’s Neutral Theory, which was  controversial as soon as it was first voiced, and has over the past decades seen sustained attacks from contradicting evidence. I believe it’s fair to say that it is, these days, a strict minority view.

@KonradRudolph I don't claim to be an expert or even remotely knowledgeable about the details of Kimura's theory, but wasn't that focusing on coding sequences? All I am saying here is that the vast majority of mutations will not cause a phenotypic effect and that isn't remotely controversial. Am I missing something?

I'm likewise no expert but neutral theory has definitely be applied to non-coding parts of the genome. Anyway, it concerns fixation of alleles in a population, not mere singular occurrence in an individual. This might be compatible with what you wrote.

@KonradRudolph yes, all I am saying here is that most mutations are unlikely to result in any changes detectable at the phenotypic level. Nothing to do with fixation, just a simple fact about how the genome is organized and how the vast majority of it doesn't code for protein so the rest, even assuming regulatory function, is far less sensitive to mutation.

I’ll add a slightly different perspective, although terdon’s answer already contains the relevant facts.
The thing that makes DNA endure in the face of imperfect copying is that, like computer storage, it’s digital. The relevant property of digital data here is that individual pieces of information aren’t given on a scale, they’re drawn from a strongly limited number of possible alternative values. That property allows error correction to take place: if possible values were continuous, error correction fundamentally wouldn’t work.
This gives the appearance of error-less copying. In reality it’s anything but, same as with computer storage and data transfer. Nevertheless, as you’ve noted, errors will accumulate, even with an error correction mechanism in place. And even though most of these errors have no influence on the outcome (due to downstream effects like the degeneracy of the genetic code), many more are detrimental than are positive. In direct contradiction of this we can see that species (but not individuals!) improve their adaptation in each generation.
Why? Because evolution. More specifically, because natural selection acts in a purifying manner to rid the gene pool of defects. This is a fundamental part of Darwin’s and Wallace’s argument. Less adapted individuals have an (ever so slightly) reduced chance at reproducing, and therefore prevent detrimental changes in the gene pool from accumulating.


text:
Question about the expression of MHC
Is it possible to express both the MHC class-1 and MHC class-2 both in one cell?

B-lymphocytes and Antigen presenting cells, both have MHC-I and MHC-II. See wikipedia page on them

could u pls explain it? or give me the link where i could get the proper answer?(in details)

the question is a little short - the comment seems to cover it.  maybe expand the question?  good question -> good answer

see this

it is clear to me now that both can express in one cell... but is it possible to express both MHC-1 and MHC-2 on same cell at the same time? If yes then how they do function?

Sure generally any cell that expresses MHCII will also express MHCI. Most cells in the body express MHCI as a self-identifier and to present intracellular proteins to T cells as a sort of a status update i.e. the cell will constantly be presenting peptides (pieces of endogenous proteins) on its surface bound to MHCI, if the cell becomes infected it could also present viral proteins on its surface with MHCI, which can potentially be recognized by a T cell specific for that particular epitope. B cells and antigen presenting cells such as dendritic cells and macrophages express MHCII but also need to be able to be identified as self through their basal level of MHCI presentation of self-peptides. The two MHC molecules are recognized by different cell types, MHCI by CD8 T cells and MHCII by CD4 T cells. So you could consider them to work independently from one another. The source of antigens (peptides here) and process for presenting them via MHCI and MHCII are somewhat different from one another.

You should find a mechanism to make this possible - mechanism that controls the ignition of the expression of MHC-I and MHC-II at the same time.
In other words, parallelism is required by the cell.
Some think such parallelism exists in one process in childbirth, for instance.
I personally think that such processes are in series, than exact parallelism, because such machinery would require large amount of memory, which our cells do not have, and fine tuning machinery, which is would require too much energy to keep to maintain the control system.
In reality, the processes seem to be happening at the same time, because there is so many of them.
However, I think this is a paralysis, since different processes are occurring.
But to show that two processes happen in exactly the same time would require the fine tuning machinery which should be found first, and also the energy aspect would require too much energy to control it.
Third argument against such parallelism is in probability theory.
I take an example about the logic behind how to falsify your statement.
Statement: If something is a B-cell, then it can express MHC-I and MHC-II at exactly the same time.
Contraposition of this: This something cannot express MHC-I or MHC-II at exactly the same time, then it is not a B-cell. This is wrong because this something is still B-cell, although it cannot express MHC-I or MHC-II in all situations.
Another perspective, the probability that these two events happen at the same time in time space with one independent system is zero.
The probability that these two events happen at the same time in time space with two independent systems is zero still. You need to have three systems to make this possible: fine tuning machine and the machines that expresses this. However, I have not seen such machinery to exist.
Generally, one point in probability space is always zero.