9

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 ...


9

As you may have realized, crystal violet can be replaced by a lot of dyes since the ethanol will wash out the dye from the gram negative cells. Methylene blue is a nice choice - malachite green may also work. A weak concentration of hydrochloric acid (3%) is also a possible replacement for the ethanol. A 3% HCl solution is used in the acid-fast stain but it ...


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

According to the sigma document you referenced (emphasis added by me): "The free acid is soluble at 0.24 g/L in water at 15ºC, while the sodium salt is soluble at >333 g/L in water at 15ºC. Therefore, pH control is very important to the use of this product. Aqueous solutions precipitate as the pH is lowered to 5." According to the FDA document ...


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

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

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 (freeze ...


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 ...


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 ...


4

It looks like Merck have different levels of quality control for their products. MQ 100 being the lowest up to MQ 600 being the highest. From what I can see the MQ200 product here has defined composition (of salt, tryptone and yeast extract in g/L) whereas the cheaper MQ100 product does not appear to have its composition ensured. I am sure your cultures will ...


3

The answer depends on the application and level of oversight/compliance you require. Most research labs I've seen use de-ionized or even reverse-osmosis water (from a tap) for most culture media applications. But when I was doing trace-metal analysis, I had to use higher grade water for almost all steps in my experiment. Mostly, we needed a certification ...


3

Yes, distilled water would be absolutely fine for your needs. You really don't need ultrapure water except for the most sensitive of applications. You also don't need to autoclave the distilled water before making things like media/buffer/etc; instead you would autoclave/filter them after you make them. You can take a look at this document for an overview ...


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.


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

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

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

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

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

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

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 ...


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

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

There is a degree of variability in milk, and in some cases this cause problems in protein work. Despite being a standard for ELISAs, it's not known to be the most reliable blocking buffer. As far as cryoprotection goes, variability doesn't seem to be a major concern. Standardization varies depending on manufacturer. The Sigma link you included reports a ...


1

This answer only concerns Streptococcus pneumoniae. The lab I worked in used cheap ingredients, the same as everyone else claimed to in this field. The only shocking thing I found was that the sterile media (already prepared) that I was given to use for DNA extraction did not work (DNA would be fragmented). It turned out the media had been waiting in the ...


1

Technically agar has first been used (and still is) in the kitchen and then adopted to the lab. In the 19th century, gelatin was used in the microbial laboratory. The problem is that gelatin is much less thermostable than agar and at higher cultivation temperatures plates will melt and get liquid again. In 1882 the german microbiologistt Walther Hesse (at ...


1

Firstly, may I suggest 2D electrophoresis as a technique for identifying differences in protein/marker expression levels. This technique will give you greater resolution and ease of comparing your sample in basically one step/experiment. Added bonus once you determine your protein of interest you can easily excise it and send for mass spec analysis to ...


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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