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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 ...


9

Always keep the raw data files! This is always a good idea for scientific data, and the only exception should be if the raw data is prohibitively large and it is not feasible to store it completely. This is not the case for gel images, so I would always keep the originals, and then use a cropped and edited copy in a suitable format for documents. The ...


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 ...


8

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 ...


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

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

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

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 ...


7

Yes, this is possible (and I have done it uncounted number of times) - the method is called "Freeze and squeeze". What you basically do is to run the gel, cut out the band of interest (be careful with the UV light, it causes damage to your DNA and also sunburns, so wear appropriate shielding for your face), dissolve it in a buffer, then freeze it in liquid ...


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 ...


6

There is one simple reason for that, your agarose gel is most likely too dense. Depending in the type of agarose, I would prepare a 0.5-0.6% gel at maximum. Synbio gives this list for "standard" agarose, which fits pretty good with my experiences. If you use low melting agarose, this table looks a bit different, as the gel matrix is not a dense. The ...


6

You have two possibilities: When you only need a quick check if your RNA is ok and you indeed only get one band, you can try a "quick and dirty" method. Heat the sample for 5 minutes at 65°C and then immediately cool it down in an icebath and keep it there until loading. By doing so, you melt up the secondary structure of the RNA and keep it in this state. ...


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

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 ...


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

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 ...


5

What I can quickly think of is that you were running the gel incorrectly: instead of from - to + direction, You ran it the other way around and your DNA went out of the gel. Another thing might be that the UV light was not turned on or is broken.


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

The instrument you are looking for is probably an UV-meter.


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

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 ...


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

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 ...


4

I have had good experience using a lithium boric acid buffer from Faster Better Media. I use it for RNA gels, but it's advertised for DNA gels. I don't think it can do protein, but I've never tried it. I'm not an electrician, but higher conductivity may be the opposite of what you want. The lithium boric acid buffer claims to have less conductivity than a ...


4

The question which buffer for DNA is better is quite old. Both have their pros and cons and I list a few of them: TBE is a better conductor and is thus less prone for overheating the gel Borate is a powerful enzyme inhibitor, so if you want to apply enzymatic steps downstream, TAE is the better choice TAE gives a better resolution for large fragments TBE ...


4

Grossly, it does not matter what buffer you use. It is the pH that matters. For DNA electrophoresis EDTA is added in order to chelate divalent cations that serve as cofactors for nucleases. Tris is the base of the buffer and is used to set pH. Along with Tris one can use Boric acid, Acetic acid or phosphoric acid for adjusting the pH. The buffering range ...


4

There are already many great answers to your question, however I thought I put my comments in form of an answer. The standard for DNA agarose gel is TAE and for the protein, it depends on the size of the protein and the gel type used! Some times MOPS works best and sometimes Tris-acetate works best. It really depends on the gel used and also the protein and ...


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 ...



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