15

The answer to the first part of your question can be found on Wikipedia: Agar is a heterogeneous mixture of two classes of polysaccharide: agaropectin and agarose. Although both polysaccharide classes share the same galactose-based backbone, agaropectin is heavily modified with acidic side-groups, such as sulfate and pyruvate. The neutral charge ...


8

1x is the final working concentration of the solution (it could be anything depending on the type of the solution). Stock solutions are made at a higher concentration; if it is 10 times more concentrated than the working solution then it is 10x. Why 50x TAE but 10x TBE? The salts in TBE precipitate at higher concentrations. In other words, the salts in ...


8

The reason fetal bovine serum (FBS) is used is because it supports a wide variety of cells and really supports their growth. It has a high concentration of growth factors, but also a low concentration of antibodies. So it promotes growth in almost any cell or tissue type without also having antibody reactions with it. Adult cows blood will have the ...


6

Although both names are quite common, both are wrong. In the original paper from 1951 Bertani was studying the lysogeny in E.coli, hence he called his media for this purpose "Lysogeny Broth" or LB in short. In the subsequent decades this name was transformed to "Luria Bertani" or "Luria Broth", which is incorrect. See the references 1 and 2 for more details. ...


5

DNA in pure water. The only time that nucleic acids would encounter pure water would be in a laboratory setting--for example after an oligonucleotide is synthesized in vitro, the protecting groups are removed from the reactive atoms in the finished sequence and the final product is cleaved from the supporting matrix. At that point you can lyophilize (...


5

In my experience, half an hour at room temperature will make absolutely no difference. Boehringer Mannheim (now part of Roche), who at one time supplied the best NADPH, used to recommend storage at 4o C. By A340 callibration, NAD(P)H is typically about 85% 'pure' based on dry weight measurements, and Sigma will try to tell you that the remainder is mainly ...


5

Yes, you can do this. As long as the final mix has the proper concentrations of everything, its fine. Just make sure you compensate the by adding 30 uL (the extra volume) less of water to the master mix.


5

The cited article describes effects of Tris on the outer membrane of E. coli. Like all Gram negatives E. coli has an inner (or cytoplasmic) membrane which is a typical phospholipid bilayer membrane containing many membrane proteins including transporters and, of course, an electron transport chain. Surrounding this there is a layer of peptidoglycan, and then ...


4

It's hard to know what RNaseZap does since the ingredients list is a trade secret. However, I expect it is a lot more than just detergent. RNaseA is extremely hard to destroy; moreover it can easily renature once the denaturant is removed. Therefore, very minute quantities are sufficient to annihilate RNA experiments. The historical method of purification ...


4

You could measure OD at 340nm. If OD340 is much lower than expected, NADPH is oxidized and does not have much biochemical activity. http://www.bmglabtech.com/media/35216/1043734.pdf


4

Thiol-Based Reducing Agents The instability of thiol-based reducing agents in solution is due to their propensity to form disulfide bonds by the following half reaction: $$\ce{2RSH -> RSSR + 2H+ + 2e-}$$ It is clear that disulfide formation requires: Deprotonation of the thiols to form thiolates (RS-). An electron acceptor. Therefore, solutions of ...


3

A "bubble" per se would do no harm unless you have cells (without walls like mammalian cells) in the suspension. If you agitate the protein mixture vigourously then it may lead to denaturation of proteins by extensive intermolecular collisions. The "froth" formation is an indication of denaturation as denatured proteins stabilize these foams [1, 2]. ...


3

I think when they sent it to you without dry ice, it is probably OK to store it at room temperature. Sigma seems to advise on their NADPH to store dehydrated NADPH at room temperature, while they advise to store the hydrated forms at -20oC. Likely the presence or absence of molecular water in the material is crucial for its storage. disclaimer: I am not ...


3

Autoclaving is the best way for sterilization. If your growth medium is large, how much chemical disinfectant do you think you would need to bring up to its working concentration? Add some acid to your waste medium (so that it's only weakly acidic), not as disinfectant, but to suppress ammonia formation. Urea will still break down during autoclave, but ...


3

Storage conditions and shelf life are some of the things that you should consider in comparing columns.


3

Extracellular matrix (ECM) fluoresces, especially Collagen and Laminin. The maximum is in the DAPI and FITC channels and the fluorescence becomes weaker towards longer wavelengths. However, since the coat on the TC flasks is very thin, I would not expect this to be a problem. The best thing is just to try it. There is also a quite famous document available ...


3

Are you sure that your TBE is at the right dilution? And that you're using TBE to make the gel and also as the buffer for running? That sort of wavy line looks like what you might get if you'd made the gel with water instead of buffer, or run it with water instead of buffer. You could also try using TAE to make/run the gel as a sanity check.


2

SYBR green is designed to be much less carcinogenic than ethidium bromide (EtBr). All these gel dyes work by intercalating themselves into the DNA stack between the bases specifically which has a great potential for causing mutations and messing with the workings of the nucleus. My remembrance is that the SYBR and GelGreen/Red etc dyes are large and ...


2

Regardless of what protocol you use, and what the advertised efficacy of that protocol might be, in any situation like this I think the important thing to consider is: what would happen if the material taken from a re-used column was contaminated by a previous application? Can you live with the consequences of such contamination? If you are preparing DNA ...


2

Seems like you have covered the essentials here. I can't think of anything else. Since the Qiagen patent on spin prep columns ran out, these kits are very cheap - $0.40 each? In the 3 or 4 kits Ive used, they all seem to use the same protocol and about the same buffers, so there might be differences in quality or yield but if so, they are small. You ...


2

Working with hot phenolic solutions is rarely been done due to the dangers of this. Phenol needs to be handled in the fume hood anyway, at higher temperatures it gets even more volatile. So if you plan to do this, do it with extra care to avoid injury. There is one paper that describes this method for bacteria, but this should work for other samples as well:...


2

To increase storage life: after gel solidifies, dampen it with running buffer. Wrap the gel in polyvinyl chloride. Place in plastic container with a lid. Store in fridge in dark. It will last for a year as long as you re-dampen it with buffer each time you access it. Don't keep it submerged in buffer as the etbr will diffuse out. this is probably the cause ...


2

I think I have some evidence that the key factor is light. Since asking this question, I changed the buffer for fresh TAE-EtBr (same concentration as in my gels) moved my gels from a well-lit area into a closed, opaque box so that they remain in darkness. After 1 week, I ran equal amounts of my ladder in the stored gel, as well as a fresh gel. The results (...


2

I always add the Taq to the mastermix. First it makes the handling easier and it avoids pipetting steps which can cause contamination and can also be forgot. Then the enzyme is very stable and will even tolerate room temperature without problem. Since we are going to heat this 30-40 up to 95°C, so this is clearly not problematic. Since the reaction mix is ...


2

Good question. I found this reference in "Molecular Biology: A Project Approach" A phenomenon called photobleaching occurs when ethidium bromide (EtBr) -stained DNA is illuminated by ultraviolet light.... This decreased fluorescence is presumably due to the dissociation of ethidium bromide from the DNA. Ethidium bromide fluoresces when it is in a ...


2

While I have never done bacterial stock cultures from spores, I don't think it is necessary, as the standard procedure for making bacterial stocks should work without problems. For that you grow your bacteria in liquid culture until you reach the late log phase (so you have a lot of bacteria in your media), take 1 ml of the culture, mix it with 100% ...


2

It would be stressful for cells to be trypsinized 24 hours after seeding. After cells are plated, there is a lag time to start growing. Perhaps, during the lag time, physiological state of cells is stabilized. They have to express proteins digested by trypsin and display on the surface etc. Then you could disturb the state again by trypsinizing cells again ...


2

Try going even higher with the agarose concentration, as it looks like you have decent separation of the small bands and poor separation of the large bands. Also try a fresh batch of agarose. I would suggest trying 2-4% in 0.5% steps If you are running 100bp or smaller fragments, you should in the range of 4% agarose. Here is a nice example of agarose ...


2

Within some linear range, the intensity of stained DNA bands in a gel is directly proportional to their mass. By making a standard curve from bands of known mass, the mass of unknown bands can be estimated. This process is called densitometry. I wrote briefly about its use in quantifying protein bands after SDS-PAGE in this answer, but I'll go into more ...


1

Might be considered "not answering the question" but: Option 1: Depending on the type of cells you are using, you can use milder methods to detach cells. Example include Versene which is essentially PBS + EDTA. Milder still is to just use PBS that has no Mg or Ca (no chelation). Option 2: Duplicate your setup - instead of two dishes - one with drug, one ...


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