CharonY

minor correction, you cannot further reduce CO2. In CO2 the carbon is already in the reduced form. The first step where of carbon fixation and where CO2 enters is a carboxylation of Ribulose-1,5-bisphosphate. In the Calvin cycle you need energy (ATP) and reduction equivalents (NADPH) that were yielded by the light reaction.

As each incubation step is long enough to ensure specific binding, cross-contamination is usually not an issue. There are different flavours of ELISAs but for instance in sandwich ELISA you usually use a high enough concentration of the first antibody to saturate the binding capacity of the respective wells. So even if you use different antibody on the same plate one would expect that small overflows would not add to the already saturated neighbouring plates. Later one you block the free antibodies using some kind of blocking reagent and so on. There may be specific variations where more care must be given, but the standard ELISA protocols usually ensure that cross contaminations as such do not occur (or are below the detection limit).

AFAIK it is just a name. However different companies sell different buffer H with slightly different concentrations of the salts, so it is not an universal name. One hast to check each supplier for details.

Also you can use cell culture bottles. Essentially I would suggest to simply scan the sites of local manufacturers for laboratory glass or plastic wares. However, you are aware that you will have several parameters at once that affect bacterial growth? What you intend to do sounds much like deep agar shakes, which are routinely done to incubate anaerobic bacteria. Within the agar you may get an oxygen gradient and a resulting unequal growth. Also the way you puncture the agar will limit the possible spread of the bacterium. Also depending on how you apply the ABs you may get diffusion effects. And also as mentioned above how do you intend to count the bacteria? If you don't spread them but only puncture the agar you won't get countable colonies.

To clarify are you trying to do SSCP with whole chromosomal DNA?

I have to revise my statement as I have stumbled across a paper describing the linkage of such short fragments utilising PCR and bridge oligos. But as long as they use the same backbone as the organism from which they put the chromosome back into it is imo still only a mutant (DNA is DNA and in vitro amplification of it is still old news).

To be honest I do not know precisely what factors attribute to eye colour (there are papers around, but frankly it does not interest me that much). However precisely because alleles contributing to brown eyes are dominant, it is possible that your child exhibits a recessive phenotype. The reason is that you are likely heterozygous to the allele(s) contributing to eye colour, but as brown is dominant, you only get the brown phenotype. Same likely applies to your partner. We can assume the easiest case, namely that eye colour is determined by a single locus. In that case we can assume that you two got Bb (with B being the dominant allele leading to brown, and b the recessive allele leading to green eyes) as genotype. and your child bb.

Sure, and it is often done. However the results tend to be disappointing unless the structures are comparatively simple or conserved (e.g. HTH or variation thereof).

Actually one would eventually need to do proper genotyping before proceeding much further. As the genetic variation among native africans is higher than in any other ethnic group I doubt that there are many markers that can be simply associated to something fuzzy as "negro". Also, I wonder how the variability among African-Americans is as compared to native Africans (as I think the drug in question was tested in the USA?).

One has to note that this part is not a quote but likely an interpretation of the author of the article. One has to await the actual publication, but ligating short synthesized fragments does appear like an utter waste of time to me.

They usually don't. Mutations normally manifest themselves after a replication step. Example imagine the following pairing: ACGT |||| TGCA Now one point mutation occurs: ACGG ||| TGCA The G-A do not form hydrogen bonds, but after replication both daughter strands will show differences: ACGG |||| TGCC and the unmutated strand will be like the original again: ACGT |||| TGCA

Actually building tertiary structure out of the pure amino acid sequence is extremely tricky. The calculation of all the interactions within a protein require massive amounts of computational power and often the results are not the same as in nature. To date one usually has to have an experimentally resolved tertiary structure (e.g. by x-ray crystallography) before one can model a similar sequence over it.

But man, 0.40$ for a genome. That would be awesome. I'd probably sequence everything I come across

In general it is accepted that oxidative stress is involved in cell aging and especially in mitochondrial damages. However a recent publication by Ristow and colleagues in cell metabolism challenges this view. Abstract: Increasing cellular glucose uptake is a fundamental concept in treatment of type 2 diabetes, whereas nutritive calorie restriction increases life expectancy. We show here that increased glucose availability decreases Caenorhabditis elegans life span, while impaired glucose metabolism extends life expectancy by inducing mitochondrial respiration. The histone deacetylase Sir2.1 is found here to be dispensable for this phenotype, whereas disruption of aak-2, a homolog of AMP-dependent kinase (AMPK), abolishes extension of life span due to impaired glycolysis. Reduced glucose availability promotes formation of reactive oxygen species (ROS), induces catalase activity, and increases oxidative stress resistance and survival rates, altogether providing direct evidence for a hitherto hypothetical concept named mitochondrial hormesis or "mitohormesis." Accordingly, treatment of nematodes with different antioxidants and vitamins prevents extension of life span. In summary, these data indicate that glucose restriction promotes mitochondrial metabolism, causing increased ROS formation and cumulating in hormetic extension of life span, questioning current treatments of type 2 diabetes as well as the widespread use of antioxidant supplements.

Uh, not quite correct. The majority of prokaryotic DNA is contained in the nucleoid area in which the chromosome is located. Usually only high copy plasmids may have a higher DNA yield than the chromosome, but they are not that common. Even then one has to keep in mind that in any case the majority of the genes is located on the chromosome whereas plasmids usually only have accessory functions. Some larger plasmids (megaplasmids) are found to be essential, but they are also usually present as single copy. Also what about RNA? It is in the title but not in the OP?

I would assume that most BS and especially MS should have handled them before. If not ( e.g. because they graduated in ethology) their job choice would be a little bit weird. On the other hand it doesn't really take long to learn. Same as above I'd say. If you intend to join a cell lab one wold usually require at least some practical courses (usually even to have done one of the theses) in similar labs. I think this is self evident. Without proper documentation the bachelor/master thesis is essentially worthless. I assume this is tied to special techniques (like RNA protocols etc.) and is likely more a specialized case. However, how is it the case with chemists or physics guys, do they similar deficits in labs?

Wait a tick. Wouldn't dissolving a lump of iron under oxic conditions yield FeCl3? One would either have to start with Fe(II) or use a reducing agent.

In my opinion it does not qualify as artificial life at all. Essentially they mutated a chromosome to get rid of ~200 kb. Mutants are routinely constructed for a long while already (though I have to admit that my largest deletions were only around 20 kb). The major twist they did is to reinsert the mutated chromosome into the organism where it displaced the existing one (or rather they searched for those that did). However, I cannot see the fundamental differences than to mutate them in vivo (and prune the DNA down there), except for the working time. Other have curated larger sequences, though it was for extrachromosomal quasi essential genetic elements and not chromosomes. To me it reads a bit more like Venter-ish publicity stunt (although in principle the results are not uninteresting). The resulting organisms are termed mutant strains and are not considered an own species. Also note that to my knowledge it is not possible to synthesize extremely large fragments completely de novo. It is possible to amplify existing chromosomes rather easily (with special polymerases) but de novo DNA synthesizers are limited to the synthesis of ~100 bp fragments.

LOL, sorry, pine_smile, but a single run of conventional sequencers (I was refering to 454 sequencers, which are a complete new breed) will give you only ~500 bp. P. aeruginosa has 6,588,339 bps. Moreover you cannot simply sequence whole chromosomal DNA, but you got to create a DNA library first, subclone it into sequence vectors, get a decent coverage (at least 10x the genome size) assemble the sequences etc. Whole genome sequencing to detect a single point mutation is not an option, trust me on that. You can only amplify regions of interest and sequence that (don't forget to use proof-reading polymerases). But you need an idea which regions to look at. Alternatively, you can try a microarray approach (as the genome of P. aeruginosa is available) and just hybridize your mutant strain against it. However you need an array with a decent coverage (most likely you got to design an own format).

Well, which methods of mRNA quantification do you know? Also, what mechanisms do eukaryotes possess to generate different transcripts from the same gene?

So apparently one consensus is that overall there are serious deficits in mathematical knowledge in most biologists (a point that I agree to). In most biological fields one requires at least basic statistical training (which imo is quite simple if you only need to apply it and not develop new models). Though at least in the postgenomics field (keyword: bioinformatics) more advanced forms of modellings are common. I wonder whether it is just a lack of training possibilities (e.g. courses, teachers) or does it come from the student's side (lack of interest) or both? I have the feeling that it might be that the foundations (e.g. in basic stochastics) are often lacking, despite the fact that they are taught (at least when I studied). Many just learn it to pass the exam and then promptly forget about it. Later on this knowledge is lacking when advanced forms of statistics and modellings are taught. Maybe there is a too large time span between the time when one learns the basics and the time when one actually has to use it for ones own work (during diploma/master thesis)?

Did I understand it correctly that you cotransfected cells with a vector carrying the gene for the target protein and a vector with the siRNA sequence, which also carries hmGFP? In that case a simpler explanation might be that there are trans reactions between the promoters of the target protein (or more precisely, gene that codes for the target protein) and that of the hMGFP (I assume you meant that the CMV-promoter is upstream of hmGFP?). This is especially likely if the target gene/protein is also under the control of a strong promoter and enhancer elements. In that case one would expect that they outdilute each other, thus reducing MGFP as well as target protein production.

I wanted to start a new topic based on the question whether biology training of biologists is worse than that of other natural sciences. Generally I would argue that it is largely dependent on the university/institute, however the question arises, whether there are intrinsic features common to all biology student courses. Phil (thanks for the correction, Paralith) gave the following points: I would argue that it largely depends on the uni. I have been in three different (German) universities and institutes so far. In two of them we (the teachers) created our own courses from scratch and wrote our own scripts. Mine were notoriously feared as I usually set the difficulty rather high when I had small groups (OK, but then I was rather generous with marks). In the institutes generally textbook variant courses were offered, but I was allowed to set my own too. I have to add that all courses that I offered were practical courses. In the end I'd say that it is largely dependent on the uni and possibly on the system used in different countries. Until recently in Germany almost all courses were more of a practical nature (with the exception of mathematics, of course. I agree with that one. I think it has two main problems. First, many biology students chose biology because they think they can get away without using maths. Personally I would argue that in most disciplines (especially ecology!) one has to have a firm grasp on at least statistics. I would argue that a biologist needs it more than the average chemists. The second point is related to the first one, statistics (or mathematics for biologists or any equivalent courses) are often poorly visited if they are not mandatory, and even then the students tend to slack off. I regularly force my students to use statistics whenever possible, though at least Germany apparently the math requirements are getting reduced. Big error in my opinion. I would argue that it may be the same for other disciplines. Many if not all areas of science have accumulated so much information that it is necessary to specialize heavily. Physics students specializing in biophysics for instance often lack knowledge in molecular physics, although both areas have overlaps. It really depends on when the specialization begins. In most universities (in Germany) you specialize after two years of studying general biology (bachelor's equivalent) then you basically choose an area (e.g. ethology, genetics, microbiology, etc.) where more practical courses are performed (and finally you do the diploma/master thesis).

Regarding GFP, it depends, how do you want to detect the GFP? Moreover, what is the strength of the promoter controlling the gfp gene? Regarding siRNA I have not enough personal experience on this issue. I can only imagine this happening if there is a massive production if the respective siRNA. I can more easily imagine a transfer of the whole vector, though.

I am still not quite clear what the objective of the experiment is supposed to be. Do you think that radiation might cause mutations that in turn increase resistance to UV radiations? Improbable, but not impossible. However you will only end up (if you are lucky) with UV resistant strains. To demonstrate which mutations were caused is not possible within a normal project budget though (less if it is some kind of course work or undergrad project). Let's go to the suggestions: - Sequence the genome: in theory it would work, but the price tag and effort to sequence even a small bacterial genome is enormous with traditional Sanger. Newer parallel sequencing methods based e.g. on pyrosequencing are cheaper, but still cost around 15-20k$ per single run. That's about the budget for a phd student for a year. -use BLAST: BLAST is basically a quick global alignment program used to identify homologous genes/proteins. It is unsuited to detect point mutations (the most likely mutations caused by UV radiation). But regardless, as you got no idea what is going to be mutated you either have to sequence the whole genome, or pick up potential genes involved in UV protection (the P. aeruginosa genome is published), make PCR and check the products for mutations. - demonstrate that UV causes mutation: that's easy to do (as pointed out by DrDNA), though I am not sure whether that's what you want to see. My final suggestion depends on what kind of project this is (e.g. practical course, undergrad studies, etc.). You can, for instance make random mutants on a global scale using marker carrying mutations (e.g. transposons) and then select for mutant strains that become UV resistant. Using the marker you can then identify the gene that has been mutated. However chances are rather low that mutations will generate resistant strains. And it is somewhat work intensive to create a transposon bank and screen it.