12

In general, alleles don't have to be the same size. Two major examples which come to mind are the Huntingtin gene and FMR1. Huntingtin is the causative gene of Huntington's disease. In people with Huntington's, a sequence of three nucleotides is repeated. The number of repeats varies, from a low of 9 in unaffected individuals, to more than 60 in severely ...


11

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 ...


10

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 ...


10

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 ...


9

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

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 ...


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

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.


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

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. ...


6

As one attempts their daily Sisyphean challenge of uncovering a new fact there is one truism, or platitude, that informs all of our efforts at troubleshooting an unanticipated experimental outcome. This can be paraphrased as: The number one cause is pilot error. Accumulated experience of bench scientists everywhere shows that their own human errors are ...


5

First, remember that identical twins actually have the same genotype. So its not exactly true that everyone has a unique genome. 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 detected ...


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

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 ...


5

This is a really interesting question and again reminds of how ignorant we are about things as subtle as this which otherwise should have been an obvious question. First of all, although there seems to be no confusion, I'd like to clarify that we are talking of the protein being separated on electrophoresis. Hemoglobin electrophoresis classically refers to ...


5

You are correct that molecule mobility depends on the mass-to-charge ratio, and this means that different sized molecules with the same $\frac{m}{q}$ will have the same acceleration. However, the velocity of a molecule moving through a gel matrix depends on the point at which the force exerted by the electric field is in equilibrium with the frictional ...


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

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-...


4

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 the PCR ...


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 ...


4

Purification and isolation of DNA bands by cutting them from agarose gel is commonplace (Lee et al., 2012). The purification step after excision of the band gets rid of most of the EtBr and other impurities. E.g., see the websites from Isogen and Sigma-Aldrich. Reference - Lee et al. J Vis Exp (2012); 62: e3923


4

It is a trick question. Red Blood Cells (RBC) are anucleated in mammals. You are not going to be able to extract anything about chromosome 1 from RBC. By the way, if you want to know how they got from 29 alleles to 435 possible combinations (in a diploid), you just have to do 29 + 28 + 27 + ... + 1 = 435.


4

Each phosphate group has a single negative charge. Not just the terminals. The reason gel electrophoresis works so well with DNA is that charge is linearly proportional to size. The longer the fragment, the slower it moves. A double stranded DNA molecule with 100 nucleotides has a charge of -200. If it were twice as long, it would have twice as many, just as ...


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. ...


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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