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18

The reason for degassing your gels is to remove oxygen. Oxygen in the gel interferes with polymerisation, slowing it down and making it less consistent, so degassing makes it faster and more uniform. From the EncorBio SDS-PAGE protocol: Polymerization is quicker and more uniform if you degas the first three solutions for a few minutes in an Ehrlenmeyer ...


8

A list of dyes is available here and a list of dyes specific to nucleic acids is available here. I think you have two choices the SYBR Gold nucleic acid gel stain (S11494) which can be detected under UV light (not sure if it can be used with polyacrylamide gels). Your other option which can be used with polyacrylamide gels is the SYBR Green II RNA gel stain ...


7

I don't have hands on it, but I will not be surprised if supercoiled DNA migrates at different distances according to some inner topological conformation (i.e., more or less coiled AND/OR more or less nicked). Similarly, this picture highlights >8 conformations. What is run in the gel is circularized phage DNA with some degree of knotting due to the ...


7

I would think GelRed or GelGreen would be an option too. They claim to be more sensitive than EtBr and certainly less toxic (even moreso than SYBR Green). I haven't personally used them against such a small bp product though. GelRed has basically the same excitation/emission wavelengths as EtBr so no equipment change is needed. Product sheet Store link


7

The major effect of changing the redox environment of a protein is the formation or breakage of disulfide bonds. Under sufficiently reducing conditions no disulfide bridges will be present, while under oxidizing conditions your protein will form disulfide bridges if it has the ability to do so. Disulfide bridges can significantly change the tertiary ...


7

Unlike in an SDS-PAGE, where the SDS adds negative charges to all proteins, in a standard agarose gel for nucleic acids all proteins should move according to their own charge. You can get a rough idea in which direction Taq would move by getting the amino acid sequence and calculating the isoelectric point at the pH you are running your get at. It might be ...


6

Yes, it is the genomic DNA that is causing you trouble. Although a brief 1000g spin should bring it all down, the pellet is never tight and you almost always are going to pull up some gunk with the clear supernatant. A better solution is to include a quick sonication step (5-10s) before the 1000g spin. That way, the DNA is sheared and it should pellet ...


6

The question is a bit vague in some important parts, so I'll have to make a few assumptions about what the authors likely meant. RNAses are enzymes that degrade RNA. There are a few different ones that lead to different kinds of degradation. The type that you would use in an experiment like this is an RNAse that completely degrades RNA. The purpose of this ...


5

I'll answer only for SDS-Page, which is the system I am most familiar with. With a discontinuous buffer system, such as the well-known Laemmli system, resistance increases during electrophoresis, as (very mobile) chloride ions are replaced by glycinate (glycine ions). From Ohms law: Voltage (V) = Current (I) x Resistance (R) and the definition of ...


5

Blocking buffer Once the proteins in the gel have been transferred to the nitrocellulose membrane it is necessary to coat the rest of the surface of the membrane with an unrelated protein. This is necessary because all proteins will bind non-specifically to the nitrocellulose. Once the membrane has been blocked the only way that antibiody proteins (the ...


5

First, remember that identical twins actually have the same genotype. So its not exactly true that everyone has a unique genetic code. But to get at the heart of it, you're asking how I can be sure that I have a different genome than you, or even than say my brother. And moreover you're asking how these differences are obvious enough that they can be ...


4

Since there's a lot of methanol in Coomassie stain, a significant amount would probably evaporate off each time you microwave it. Therefore, it'd be probably a good idea not to reuse it because (1) you're losing methanol, and (2) the concentration of everything else is thrown off. Why do you boil the stain? I just pour mine at RT right on the gel and it ...


4

I have been running gels with different Acrylamide/Bisacrylamide ratios recently. People usually work with 1:37.5, 1:29 ratios which are commonly used for DNA and Protein gels. I have noticed that when you work with lower ratios 1:200 - 1:500, degassing becomes fundamental to guarantee reproducible resolution of my proteins. If I don't degass the mix in one ...


4

Doing westerns with primary tissue can be tough, especially because of the presence (sometimes at high levels) of extracellular matrix (ECM). This material can be quite resistant to homogenization and some lysis buffers. One method I have found to work is to snap-freeze the tissue in liquid nitrogen (not on dry ice or at -80°C) directly after removal ...


4

This is the figure the question is about. On the right is the control experiment with GTP-γS, on the left without it: The bands that are visible in both experiments are unspecific binding. If GTP-γS doesn't affect their presence, the mechanism by which they bind to the column can't be specific to the GTPase functionality. The proteins the authors were ...


4

These are sequencing gels (in the cases here even radioactive ones) - they are run a lot longer than ordinary agarose gels and they are made from polyacrylamide. Im my experience, the most likely cause for skewing of lanes (not only bands) are samples, which still contain too much salts from the PCR reactions. This can also happen to only a few bands as seen ...


4

I normally use 1-2 ul (depending on whether my gut tells me I have a lot of DNA, or a little that day) in a 70 ml 1% gel (which is probably 60-65 ml after boiling in the microwave). I have no trouble whatsoever seeing ladders and DNA samples of down to 100 ug in wells not much bigger than about 4 mm. Here are the factors that you can manipulate to alter the ...


3

I've run a test following this protocol to the letter... PCR step by step: Collect all ingredients (excluding TAQ) from the refrigerator and keep cool (on ice). Leave the TAQ in the freezer until required. Make master mix by adding all ingredients (excluding TAQ) using fresh pipette tips for each ingredient and using the volume specified in ...


3

If you tried to set up a system in which movement of DNA molecules through a gel matrix was driven by pumping buffer, the gel would become compressed. I am old enough to remember running vertical agarose gel electrophoresis overnight, and these gels would collapse under normal pressure to about 2/3rds of their initial height. A gel porous enough to give ...


3

The answer to the part of your question concerned with average lengths of restriction fragments is: if the DNA molecule being digested is of random sequence, and is 50% GC/ 50% AT, then the probability of finding any given short sequence at any position is 1/4N where N is the length of the short sequence. So, for example for a restriction enzyme with a ...


3

If the salt content or pH is different than "normal," run time can be affected. After all, you're applying an electric current to the sample(s) and ion concentrations and pH are sensitive to that current. Of these two, salt/ion levels are more likely to have an effect as your samples/markers are likely to be adequately buffered.


3

Gianpaolo is correct that you are seeing the different conformations of circular DNA. Growing plasmids up in bacteria produces 3 main forms: relaxed, coiled and super-coiled. Relaxed tends to run a little higher than the expected size because it cannot travel as efficiently through the agarase; Coiled migrates a little farther than the expected size ...


3

Possibly partly offtopic, but I'd like to add that when using dna fingerprint as evidence of identity in a legal investigation, the odds become smaller due to combination of human factors and reduced accuracy. There is the twin issue already mentioned, but in addition there are laboratory errors and the possibility of contamination with other evidence. ...


2

Well, to rationalize everyone's comments, I think @leonardo is right. This is a denaturing SDA PAGE gel. The migration of the SDS Micelles which are negatively charged, depends upon the shielding of the solution around it. The difference in mobility is because the SDS micelles will experience a slightly different field at pH ~6.2 (MES) vs 7.2 (MOPS). ...


2

SDS PAGE system rely on the fact that protein is denatured and surrounded by the SDS negatively charged detergent micelle. This eliminates most of the charge and idiosyncratic solubility differences from one protein to another and gives a reasonable separation based only on size of the protein which is related to the size of the SDS micelle around each ...


2

If you use pre-staining, the stain migrates as well in the electric field and this can lead to some parts of the gel being unstained. This would look pretty much like you describe, the ladder being invisible as well points strongly to that part of the gel just being no stained properly. It sounds a bit unlikely with your running parameters, I've only seen ...


2

The thermal cycler adjusts sample temperature based on the volume of the sample. So if you run samples of different volumes side by side, not all of them will cycle through the optimal temperatures of each step. If you were working with a "hot start" polymerase this is even more critical, as the Taq won't be able to amplify at all unless the sample is heated ...


2

not from gels but physical force can be used to separate molecules.. e.g. density centrifugation, filtration. Closest to what you are asking is size exclusion chromatography in HPLC where molecules are separated based on size and high pressure is maintained in order to pump the solution (containing the analytes) through the stationary phase. As Alan Boyd ...


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

The loading buffer you add to your samples for gel electrophoresis has a few different purposes, but the exact amount does not really matter. The purpose of the loading buffer is to make the sample heavier so it is easy to get into the pocket and stays there to visualize how far the gel has run to denature the sample (only for denaturing gels) Sample ...



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