CharonY

So what do you think do these spectra tell you?

It is a bit unfortunate that you have not diluted it more to obtain colonies. However, if they form streaks it is extremely unusual to see only black dots instead of a large brown-black zone exuding from the streaks. What do you define as spot?

Wouldn't be the laws of thermodynamics up on the list?

Dishes serve a very different purpose: sterile working and actually seeing the colonies. Also you will notice that many will not behave the way they are supposed to do (which is also part of the training). Moreover, to my limited knowledge there are only kits for a couple of well-defined groups and even so the identification rate is not stellar. Even well-established one like the enterotube hover at around 90%- again, for well characterized groups. A good microbiological training should cover all the basics and then move on to using kits. In this particular case for instance, it is important to note, how the black dots look like. Are there in the middle of the colonies or (as it supposed to be) diffusing out of the colony (essentially a zone)? Generally the reaction is quite strong with Enterococci and type D streptococci, and if actually have active growth it should be easily distinguishable. What about colony size? What is the color of the colony? Staphylococci tend to be largish white whereas e.g. corynebacteria tend to be smallish yellowish and micrococci whitish grey, each without a brown or black zone. Also regarding the MSA, where the colonies yellow (with a yellow zone) or more orange? Note that I am not primarily a microbiologist and am just saying something from memory. Apparently the training I received a decade or so back was not that bad as I can still remember some stuff.

For starters enterotubes were developed for the identification of Enterobacteriaceae. Thus if you get thrown at with random bacteria it is pretty much useless. Second, the reason for doing these plate assays is train yourself on the technique and the logic behind it and not to stay on the level of a kit user. That being said it would be helpful if you could provide a list of from which you have to select the bacterial species in question, as the combinations in question leave room for quite a number of potential species.

Essentially you are right (I think), though it would be easier if you would be a bit more organized in formulating your thoughts. Plasmid size does not figure in right now. You have the following steps starting from having the plasmid (and insert) purified: restriction, dephposphorylation, ligation, transformation. Each of the steps can be incomplete and you make controls to ensure that that particular step worked. Based on this you are correct to assume that if everything worked perfectly, the plasmids gained from b should be lower than a, but how does it compare to c? Maybe you should give it a try and answer the hypothetical situations that I gave you. Also keep the subsequent steps of a cloning experiment in mind.

You are probably thinking too complicated. Let us take a step back. What are controls for? Answer: they allow us to check what happened at every step of the experiment. Now back to the controls at hand. Non-dephosphorylated vectors can religate and therefore you are correct in assuming that normally you would have a higher number of clones transformed with non-phosphorylated vectors. However, you would also have to look longer to find a clone with the proper insert. Now imagine you conduct experiments in the op and imagine the following scenarios: 1) a has the highest amount, followed by b, then c 2) a has the highest amount, but b and c are equal (though both lower than a) 3) a, b and c are equal What happened at every step? It appears to me that you understood the individual reactions, but you just have to piece the things together. Again, remember that controls are supposed to work in a specific way, but if they don't they hint at something gone wrong.

Several mechanisms pertaining to mRNA stability are known for much longer. However the regulatory role of small RNAs (again, it is not the sole regulator) is a rather novel aspect. Thinking of the regulation as a series of defined switches is often not applicable. Generally you always have a equlibrium reaction. The level of the RISC complex (together with the status of other regulatory elements) regulate the total amount of protein being produced. Each of the circuits can and often are responsive independently from each other. Quite often you will find that a given mRNA is produced (as long as there is no transcriptional repression) and then the due to some factor (or lack of it) the mRNA gets degraded (by RISC and other processes) with only low level of protein translation. Bottom line: things are always a degree of magnitude more complicated than one thinks it is.

Actually this is a given, especially for association studies. For instance, let's say we want to investigate what causes cancer. There are virtually unlimited degrees of freedom, when it comes to the study design. We could for instance look at any type and amount of food, or excercisse, mobile phone usage, etc. In contrast, only a few of these choice actually will be true positives. Since the search space is virtually unlimited there will always be more false than true positives. Of course this is even worse if the study design itself has limitations.

The RISC complex is far from being the only system controlling mRNA stability. It is but one of many layers of regulation. In addition the regulatory cascade can be very complicated and is often not governed by the gene product of the target. They can e.g. be present to maintain certain equlibria, triggered by certain stimuli, be involved in feedback inhibition of metabolites and so on.

Fertilizers are probably less worrisome in terms of toxicity. However think about the ecological impact. E.g. algae growth, sudden biomass increase etc.

For the most part correct. The phosphorylation is indeed done to ensure that the vector really carries an insert. Of course, doing it alone does not yield much info, but it does if compared to other results. Most controls only do make sense in comparison with another experiment. So if you compare b to c what outcome do you expect (in terms of colonies), assuming all steps of the cloning experiment worked perfectly. Under which condition would that not be the case? Visualize all the steps of the experiments and then speculate.

Nope. The microRNA is part of the RISC complex. Together they they act on mRNA.

For transformation frequency the same applies. The uncut plasmid will always have a higher frequency than any ligation. How would that be a control. What info would that give you regarding your ligation?) Again, generally it is a control for the cells. In addition you can check ligation efficiency. Regarding the dephosphorylation: you are right that it prevents self-ligation. But think about it a little more. What effect would that have on the cloning? I.e. why do you dephosphorylate in the first place? Following that line of though, what would you expect when comparing b and c and what would it tell you?

If you really want to do lab work in the bioengneering area you could try to either get a technician job after either bachelor or, probably more preferable, a masters. The big question, however, is where you want to work. While genetic manipulation is all nice and good most of the time you will also need e.g. solid fermentation or similar techniques. In the end it is important to inform yourself about what work you want to do and plan your curricula appropriately.

If the pI is lower than the ph of the buffer they will be negatively charged, yes.

a) is not for finding the efficiency of the transformation, as it will have to evaluated for each construct differently. This is because the transformation efficiency is dependent on the cells as well as the construct. However it is a control for the competence of your cells. For b and c, what do you think will be the difference between those two conditions. What does the phosphatase do and how does it affect the ligation?

Moved to the homework area. Also it would help if you would just shortly copy the question in here instead of using a link. Spammers are everywhere.

Antioxidants are old news, too.

You should check whether the effect is reproducible. During evaporation CO2 and hence, buffer capacity gets lost. However, the proteins themselves are afaik not different enough to result in different pH (I would have to check the sequence to verify that, though). I would rather assume that it is essentially a problem of a non-buffered solution. Small difference of additives or even protein concentration can shift the pH easily.

There are a number of mechanisms and the majority is probably still unknown in detail. Most rely on certain gradient e.g. along developmental axes. One well-studied element is the chromatin state of Hox genes. See for instance: Howard Y. Chang, et al. Science 326, 1206 (2009), for a relatively easy to understand read on the topic.

Sustainable is missing somewhere!

Quantum solar organo femto-crystal dots.

Well evolutionary models can be derived on the gene level, which is done routinely. However, if you want to make population studies and derive for instance the chance of fixation, you will have to figure in fitness. The latter can only be achieved by measuring on the level of reproductive units, which generally means the whole organism. Even mobile genetic elements, which are as individual as possible when it comes to genes need a vehicle to persist through generations. Though of course the horizontal transfer can be measured with other metrics.

The single most important bit of advice is not to focus too much on the courses per se. In reality you will most likely already be a grad student before realizing what benefits you most. In many if not all cases there is no clear trajectory in which you can project where you will end up and what precisely you will need for a given project. A good foundation in all the basics is helpful, of course, but chances are, you will have forgotten a lot once you come to the point where you actually seriously need it. Having a good basis makes it easier to catch up, though. Finally the most important bit is to realize that grad school is not an end to itself. You will ask yourself where do you want to be in the end? Academia? Why? Or industry? Why? What other alternative? The PhD can be an entry ticket for something, but unless you just study pro arte, you should be sure that you are moving in the right direction.