CharonY

ZING!

 

http://news.bbc.co.uk/1/hi/technology/4530930.stm

i dont make adjustments, but im a huge fan of it. it's not research in a can, but its proven to be a good first stop just to overview and browse by hyperlinks for me.

I still thinkt that only means that the Encyclopedia Britannica is, despite its name, not very good either.

Mostly I only correct gross errors in Wikipedia, too.

Given the mindset of many scientists that I know, the younger ones won't acknowledge anyone better than themselves, older ones do not acknowledge the existance of competitors ;P

Actuallly waterin the gels has some merits. There are number of coomassie staining protocols and in most of them siginifcant shrinking of the gel occurs during fixation and staining. Those gels easily crack if you dry it, especially if the ambient air is also dry (in deserts, of course, but most labs have a dry air due to their ventilation systems).

Regarding storage, if you use water I fond that I still could use mass-spec on most gels even after half an year or longer, though mould could be problem till then. In fact, you can also restain your gel several times, if need be.

Well somebody surely does know something. Yet it might be of advantage if one could specify what one might want to discuss (or ask).

Based on different sources.

No, I meant per what unit. Do you get less DNA for example from 1g Broccoli as compared to 1g Banana?

There are a lot of reasons and one question is to what you have standardized your yield.

Common examples are different effectiveness of cell disruption, higher weight of the tissue (e.g. water content as you mentioned) and so on.

The basic thing to keep in mind is that you usually only have one copy of the genome per cell. Only if you disrupt the same amount of cells you will get equal yields.

Wow, this would be a very special interpretation of the question. There are a number of plant that have a anaerobic phase, but always connected with aerobic respiration phase. If that is indeed the right answer, I think one should smack the one writing that question.

I am not really a plants guy (unless they get infected), and I cannot think of a single anaerobe plant. However, you are right in your suspicion regarding fungi. They are clearly not plants.

In addition, during photosynthesis oxygen is produced and thus no true anoxic conditions would be there...

I am all for a Science City that was harsh barren and cold, deviod of all human touch.

What the heck would that have to do with science? Moreover In case you didn't notice, scientists are all humans, too.

Ohter than that at least in Singapore and Japan science cities have been established. Although I am quite sure it was not what you had in mind...

Sounds like homework. Lookup what a DNA polymerase is doing (hint: PCR). I assume here that the DNA dependent DNA polymerase is meant (instead of the viral RNA dependent one). The second half sounds incomplete.

Well, I agree with the first part, but have to disagree with your assessment of photometers. Those that allow wavelength scans also allow an (rough) estimate of contaminations (in many protocols RNAse steps are involved anyway, protein contamination might be important). And regarding the amount, with special cuvettes you can use very low amounts of DNA. With the usage of newer machines (I like the NanoDrop for instance), you can go down to 1 µl of volume and few ng of amount for measurements.

Are you seperating whole genome DNA?

Usually one need pulse field gel-electrophoresis for seperating DNA of that size.

Actually one usually quantifies DNA with a spectralphotometer. On gels one can use mass markers and densitometric measurement. I am not sure how good that works for chromosomes, though.

Also note that selection occurs on the phenotype and not on the genotype level. Most genetic diseases are the result of recessive alleles. And recessive alleles will usually not be eleminitated from the gene pool as there are usually heterocygous carriers of these alleles which do not express the disease.

The selective advantage or disadvantage (aka fitness modification)is conferred to the whole organism, as melanocytes cannot propagate outside the organism.

You already got the right answer. To make it short:

bacteria possess certain restriction enzymes that degrade external DNA coming into the cell. In order to avoid restriction of own DNA they methylate it with a specific pattern that is recognized by the restriction system (which sometimes is part of the methlyation system) and restriction is avoided. DH5alpha has one major mutation in one of the restriction systems (hsdR, I think), that methylates (together with other subunits) own DNA and degrades unmethylated one. If you introduce unmethylated DNA in it, it won't get degraded.

DH10B got some more mutations in restriction systems (mcr-system, if memory serves) that can regognize methylated foreign DNA and degrades it. Thus in DH10B you can also insert DNA that was methylated (as you stated).

However, there is also DH5alpha MCR, that carries the same mutation, so it can also be used for methylated DNA.

Actually no. The answer to your question is the first part, both methods use different properties, regardless of detergent. SDS PAGE does not separate by mass, but by its form which becomes roughly porpotionate to its mass due to denaturation.

You can also seperate native proteins (as in blue native gels), and you will also find that larger proteins tend to run slower than smaller ones. Here however globular proteins will tend to be faster than linear ones with the same mass. In PAGEs proteins are retained by the pores (thus larger proteins are slower), whereas during equilibrium centrifugation they are moved towards the bottom until stopped by difussion forces. Proteins of a higher mass require higher diffusion forces to move up again (or rather have lower diffusion forces counteracting the centrifugal forces) and thus penetrate deeper into the centrifugation vial.

cause Bacteria's have ability to mutate quickly and somehow it will develop an enzyme to destroy the antibody.

Not quite correct. They mutate (largely) independently on the presence of antibiotics. Antiobiotics only select for the already resistant ones.

Well.. different yield based on what? Weight of the sample? Different cells for instance have different weight to DNA ratios.

Or some cells/tissues are harder to disrupt (and so on and so forth).

I would like to know how come when we separate proteins in velocity sedimentation centrifugation (i.e using a sucrose gradient), larger proteins move faster than smaller proteins, but, if you separate proteins in SDS-PAGE, larger proteins move slower than smaller proteins.

 

If we would have tried to separate proteins which were treated with SDS, by velocity sedimentation, would the larger protein move slower than the smaller, like in SDS-PAGE ?.

 

My hunch is that if you talk about globular proteins, then the larger the protein, the faster it goes through the gel, but if you talk about filamentous proteins (SDS causes all proteins to become that way), then the larger the protein, the slower it goes through the gel, because friction now plays a much bigger part. I'm not sure if I'm right though...

In principle you are not wrong, though you compare apples with oranges in some parts.

The point is that with each method depends on a different property of the protein. In SDS-Pages the size of the protein determines the runtime and with SDS (in addition to adding the needed charge) the proteins are denatured. However you do not really measure the mass, but just compare the run time of the given proteins with markers, which might or might not behave as the analyte.

In contrast, the analytical ultracentrifugation allows direct calcualtion of the mass. For instance with sedimentation equilibrium one can directly determine the buoyant molecular mass. Adding SDS here will mainly will directly lead to an increase in the mass of the protein. The shape usually does not an high impact on subsequent calculations here.

The buffer does not depend so much on the dye but rather on the polymerase that you use. Most polymerase buffer consist of a basic Tris-buffer (ph 8-8.8), Mg, and a mix of KCl and (NH4)2SO4. Other ingredients (e.g. BSA, Triton X-100, betaine etc. are possible but not essential).

In short, if you use your own polymerase, just add the buffer for that enzyme.

Hi everyone. I want to do an experiment on the growth of a certain kind of bacteria in acidic and basic environments. I have 2 questions:

 

1. How do I obtain a pure culture of just one kind of bacteria, without any contamination (preferably a non-pathogenic bacteria)? Do I need a sterilized lab for this?

In addition to sterile techniques (in the easiest case you can work in the vicinity of on open flame to reduce contamination) you need a means to sterilize your medium. Moreover, you should try a medium that reduces the risk of getting pathogens. Avoid too rich media as well as media that selects for pathogens (e.g. brain heart infusion, blood agar). As mentioned above you should make agar dilutions (streak dilutions, get single colony, streak again, repeat), for anaerobes you can also do deep agar shakes.

However, as mentioned above, with unknown bacteria you can easily enrich pathogens.

2. How do I go about setting up the acidic and basic environments? I have pre-prepared agar plates at school that I will use. Do I just dump acid or base all over it? Or do I use some other way (like using the little discs of filter paper)?

 

Thanks and excuse my ignorance. I know NOTHING about this.

To test for acid/base sensitivity you can test with the filters, but then you already need an isolated bacterium for Ph you have to check what buffer is in the medium. LB is an often used medium that is essentially only buffered by the tryptone. Problem with just dumping acid or base is that the reached ph might be very unstable. For this, you should add a buffer with a fitting pka. You might e.g use bis-tris for slight acidic and tris for basic media. Hoiwever, many buffers are toxic to microorganisms in too high concentrations. Especially for soil bacteria carbonate buffers are more physiological.

I'll assume that the injunction against loans doesn't apply to scientific grants, since they're used for research and not personal gain.

s for myself, I'd keep doing research, hopefully in a faculty position (hey, who'd turn down a faculty member they don't have to pay?).

*Sigh* Heaven. Or maybe a pure research institute given sufficient funding. *sigh once more*

One Dalton is 1.65 x 10^-24 grams. Actually that is all you need to calculate the rest.

I am not perfectly sure what you are asking. Using these primers will give you a single product starting from 984 and ending with bp 1401 of the template.

I assume you wonder whether everything within a cell decomposes once the cell is dead? Answer is: not really. It depends on the stability of the compounds. A living cell retains a certain intracellular environment which stabilizes for instance enzymes or nucleic acid. Once the cell is dead the homeostasis is no longer stable and some molecules degrade faster than in a living cell (e.g. mRNA).

For the second part of your question: I have not the slightest idea what you might be talking about.

I just hope that that was not serious.