CharonY

Well you need a medium with agar with it. But two caveats: a number of mouth bacteria are potentially pathogenic. So I would not advise to use media in which they can propagate. I probably would not want to do the experiment with younger kids, actually. Second: unless you brushed teeth just before sampling, the amount will not be significantly different between humans and dogs. Expect in the area of 10^9 bacteria per ml of saliva (so dilutions are necessary to count the colonies). And yes, definitely wrong forum section.

I assume this is just a "training" proposal? Usually you first to have to make sure that basic equipment for handling is present P. falciparum, as well as labspace (e.g. you need at least a BS2 lab). Most project grants do not cover basic equipments. Before I comment on the consumables I would advise you to look at existing protocols for the manipulation and maintenance of Plasmodium cells, though. You need to know this before you can write the grant. Check the malaria journal (it is open access) as well as some of the parasitology journals for this. I recall a paper about the assessment of different deletion protocols for P. falciparum around last year or so in the malaria journal. For standard cloning techniques check out "Molecular cloning" (Sambrook and Russell). There are tons of information in it and I recommend it to everyone. If you got a basic idea of the experiments you want to do, you can start checking out prices. Mutagensis kits are, btw. not essential. What they do is creating a mutated insert. Most of the time you can do it just with the right primers for less money. In order to get an idea how much the kits cost, I'd advise you to browse at least to Promega, Qiagen, Invitrogen, as well as general lab equipment vendors (e.g. VWR and Fisher).

Well, these are two issues. Basically if the data (analysis) is not of sufficient quality one cannot declare an effect as busted. This problem does not go away if you just do something else with the same lack of rigor. Also they usually do not really produce an awful lot of data. Usually there are only two to three data points per experiment. Much of the fun is not in the results but the whacky way they try to approach it. Also the replicates are quite often not really replicates in the strictest sense and so on. Of course there are limitations due to time and cost and it is an entertainment show after all. However, sometimes they do use the word "science" or "scientifically" quite a bit too often for my taste.

Also what they do as replicates are usually not statistically significant. It is often fun, though. Maybe a bit like CSI for the forensic community, only better. Well, Zombie Feynman is quite convincing, though.

Afaik there is almost nothing definite about them. Just to give an example I checked the same journal as the link above (PloS Path.) and found this: Link From the abstract: "Our results definitively ruled out the existence of “nanobacteria” as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call “nanons.” The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor." Well, OK according to these authors they are definitely not living. I have not checked into this topic seriously, though. I would be interested to look at some samples under the AFM myself, though. The report is quite convincing, btw.

Hello, you may have a problem there. The only methylation system (E. coli has several) that interferes with XbaI is Dam. Unfortunately I belive that DH10b are dam+. So if you isolate DNA out of DH10b chances are that it won't cut. So if you are dependent on that particular restriction site you can either transform a dam- E. coli strain with the vector and then re-isolate it from it. Alternatively you can try to look for an isoschizomer that is not methylation-sensitive. I do not know one out of my head, though.

Actually I have to complicate matters by adding that occasionally paternal transmission of mt DNA in animal, including humans, occurs. For instance: Schwartz M, Vissing J (2002). Paternal inheritance of mitochondrial DNA. N Engl J Med 22: 576–580

Trouble with this is that we would have to distinguish living from non-living a priori. Which is the basic problem I have when it comes to the definition of life. Edit, I missed that one:

In Germany you used to have work on your own on a science project (often a smaller part in an ongoing research effort) for around 6-9 months in the lab to get your degree in the last year in chemistry (and physics and biology). As there were no bachelors degrees that was the only degree (diploma) you can get (and had to get if you wanted to continue to a phd).

Is the calibration consistently off? Possibilities: array is not correctly installed, or it may be somehow damaged. Try reinstalling it again. If it does not work I'd suggest calling the support.

I have to edit my post, as I was referring to the wrong PloS journal. However, quite a number of papers are being rejected even in PloS one due to the same reasons as in other papers. The things, the editor's name gets published, but not necessarily the other referee's he/she has been consulting. Lack of importance (or lack of novelty) is quite a common reason not to publish something for basically all journals.

Depending on the field you might experience that any jobs are in fact not directly involved in research per se. Other job for phDs include product management, sales/support, consulting and so on. In numbers, at least for biologists, the average salary for a postdoc is at around 30000-35000$ annually in academic jobs. This varies depending on field, country and experience, of course. These jobs are usually time limited, though. Faculty jobs are higher (often around 50-90 k, depending on position), but getting one is not easy and it takes quite some time. I think most score full positions when they are around 40ish at the earliest. Chemists and physicists usually have a higher average salary, though. Jobs in the industry (e.g. as technical consultant or similar) often start at around 60000$. ...

That would not pose a problem for anaerobic respiration, though.

You generally use polyclonal antibodies as second ones. These happen to be IgGs

That's rather unlikely. Just by browsing I do not see an awful amount of paper on whales and many of them are using molecular biological techniques. In other words, you only need a couple of cells to do the respective experiments, slaughtering a whole whale is, in the truest sense of the word, "overkill". Also apparently a lot of the meat that is not getting eaten simply lie rotting around: http://www.news.com.au/adelaidenow/story/0,22606,23206289-5006301,00.html Finally you may want to read the following article: Parsons et al 2006, Marine Pollution Bulletin It’s not just poor science – Japan’s “scientific” whaling may be a human health risk too

Yes, that's why it is important to include statistics

So do you mean that you made a run with various concentrations on a single day and then repeated the whole assay on another (i.e. there is not likely a bias towards specific concentrations?). Regarding the analysis, "the error bars are pretty tight" is at best just a qualitative assessment. At the very least do a t-test.

Before you proceed thinking in that direction have you double checked that your results are statistically relevant? Moreover are the results repeatable? Was everything standardized? That is, where the experiments done in one go, or on different days? Where the cells from a single split culture or could they have different histories? Whenever something counterintuitive pops up it makes sense to be really really sure that the effects were not due to handling, different batches of cells etc. Otherwise you are going to lose a bunch of time.

Well, basically it is not uncommon to have concentration dependent effects. It is kind of strange that a high amount has seemingly beneficial effects, though. In general, concentration-dependent effects can be unspecific. E.g. w/o knowing details one can speculate that the redox enzymes decompose and increase the amount of copper in the medium. However, I'd not assume that this would be beneficial (an iron enzyme might make more sense, depending on the cell culture). One question would be how this bacterial proteins is supposed to affect the growth of the cell line in this particular experiment?

Unfortunately there are a gazillion possibilities why it does not work. First question is whether there is production of the correct protein at all. Based on the OP I'd assume that there was low production. Have you checked whether it is the correct protein (e.g. by MS/MS)?

As I have mentioned above, rRNA is often isolated by microbiologists interested in the active microorganisms in the environment. RNA degrades much faster than DNA, so if you only go for the DNA chances are that you will also have many sequences of bacteria that are long dead. Everyone amplifying rRNA sequences will at some points notice that: if you do not work in a clean area you might get amplifications using universal rRNA gene primers even if no template is added (one of the reasons why these bothersome PCR cabinets are used). If you isolate RNA on the other hand, chances are higher that you will get sill living organisms. In other words, you will get somewhat different results if you amplify directly from DNA as opposed to RT-rRNA. This is of course only the case in complex communities. If you got some highly enriched samples or even pure cultures it does not make sense to go for the RNA.

It could very well be a respect thing. I noticed that the staff here (in Arizona) always refer to the professors as Dr. something, whereas postdocs are addressed without a title, for instance.

Actually you are right to consider how the antibodies were produced in the first place. It is e.g. a difference if the protein or only the relevant peptides were presented (or if the design was first done in silico). I assume that in this case the antibodies in question have never before been used in a Western? Of course one can try doing a non-denaturing gel, though the resolution would be rather bad. Anyway, APCs do not denature proteins (as antigens) in a similar way as SDS does, but they degrade it. So in the end they would present peptides. A discontinuous epitope of that antigen has to remain, per definitionem, intact. Otherwise it would not be an epitope, nor would it be considered discontinuous.

To vbreemars, just to clarify: 27F and 1492R are universal primers, these are conserved regions of the 16s rRNA gene. If they were specific primers you would only get amplificates from a single genus (or even species). Anyway, as I mentioned above, both approaches are viable. RNA has a faster turnover, though, so microbes detected that way are more likely to be still active. What some do is to do both, used DNA and cDNA to analyse the differences between those two pools.

I am not sure what your question is. Proteins of the Lrp-family bind DNA via a N-terminal HTH motif (pretty much standard in that regard). Generally, if you find that a particular paper is missing information that you need, try to track down a review. There are a couple around of LRP-type proteins. If you are interested what the Lrp-protein in E. coli does, there are number around describing its regulon (it is rather a global regulator IIRC). Or are you actually interested in the particular mode of the Lrp-regulated Pap-pili switch? That is, how methylation is inhibited?