12 votes
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Are alleles equally sized?

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 ...
  • 1,544
6 votes

What is possibly wrong with my gel electrophoresis when i don't see bands of DNA ladder?

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. ...
  • 3,432
5 votes

Why does hemoglobin electrophoresis in sickle cell trait gives two bands but not three?

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 ...
  • 3,647
5 votes
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How does SDS-PAGE separate based on mass?

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 ...
  • 8,179
4 votes

How bad is ethidium bromide in your plasmid for downstream applications?

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 ...
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4 votes
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Why would I see no bands in DNA gel electrophoresis of red blood cells?

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 ...
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4 votes
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Are longer and shorter DNA similarly charged?

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 ...
  • 7,627
4 votes

Why do you have to submerge your gel in buffer before adding DNA

This has a very simple reason: Adding the buffer later poses the risk to wash out your sample from the well as well as mixing samples. There is another reason: It makes loading easier. The sample is ...
  • 50.6k
3 votes
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Why does my gel have such poor resolution?

Although your tech is referring to your DNA samples, your ladder also indicates there is some room for improvement of your running technique. -20C for a week should not have impacted the quality of ...
  • 344
3 votes

Agarose gel Ladder smear

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 ...
  • 41
3 votes

Quantifying DNA in a band on gel electrophoresis

All three methods could be used to measure the amount of DNA. However in practice, method 2 (estimation by dye brightness) typically works best in a normal workflow. It really depends on what you plan ...
  • 344
3 votes
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What do we call this adjustable platform used to ensure that something is positioned strictly level in a lab?

I remember using those for agarose gels as an undergraduate, though using them for PAGE doesn't make a lot of sense unless your bench is seriously out of level. Anyways, they can be called levelling ...
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3 votes
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Protocol to dilute DNA step ladder?

You'll probably have to titrate it down yourself. You might be able to estimate: The detection limit for ethidium bromide staining is about $1 ng$ per band. The insert says it's at "1 $\mu g/ \mu l$", ...
  • 196
3 votes
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Long term storage of agarose-ethidium bromide gels that have already been cast

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 ...
  • 3,675
3 votes

Why is gel used in electrophoresis?

In gel electrophoresis, the gel is the mechanism by which macromolecules of different sizes are separated. By loading the gel with amino acids (or proteins or DNA), you start all of the samples ...
  • 111
3 votes
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What is meant by 4 –12% or 8% SDS-PAGE?

Do the percentage values refer to the percentage of acrylamide in the gel? Yes. The 8% gel is 8 g acrylamide per 100 mL. The “4-12%” gel is a gradient gel, which are useful for separating proteins ...
  • 8,179
3 votes
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Electrodes in non-conducting fluid? Are they neutral? Electrolysis, Electrophoresis

Electrodes are neutral, but they are held at different potential. This is similar to the current flow in a wire: although current flows, the wire remains neutral, i.e., no charge is accumulated (...
  • 3,802
2 votes

How can I purify RNA after gel electrophoresis to remove residual acrylamide?

If you don't want acrylamide in your preparation, don't use it, as you will always have some carry-over in the solution. And you will not get rid of it completely, as it at least partly co-...
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2 votes
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Alternative protocol for evaluating RNA integrity, using a bleach gel

I don't think bleach can denature RNA. Bleach is an oxidizing agent and it will damage the RNA. Also, the protocol mentions addition of hypochlorite before heating which I think is illogical because ...
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2 votes

Long term storage of agarose-ethidium bromide gels that have already been cast

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 ...
  • 4,470
2 votes
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Freezing after restriction digest

From my experience I would say that it is no problem to freeze the digested DNA, when the enzyme is inactivated (even when not, for this time period I wouldn't expect much damage). However, if ...
  • 50.6k
2 votes

Why does my anti-ubiquitin antibody visualization not work on my PAGE gel?

There are stable ubiquitinated proteins in mammalian cell lysates even if active proteosomes exist in cells. First, you might want to make sure that the antibody is applicable to WB. Then, you ...
  • 1,116
2 votes

What are the pros & cons of site-directed mutagenesis? What are the alternatives?

The 'Quickchange' method is probably what you want. If you are worried about self complementary (which you shouldn't be too much) you can actually stagger the primers so they are not fully ...
  • 1,415
2 votes
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What are the pros & cons of site-directed mutagenesis? What are the alternatives?

SDM will be the perfect tool for this mission, unless the plasmid in question is very large (20+ kb). When you have performed the SDM, digest the products with dpnI to remove the original plasmids, ...
2 votes

What are the possible reasons to get extra unrecognized band in agarose gel electrophoresis?

Looks like genomic DNA contamination to me. More importantly, does it really matter? I certainly appreciate the curiosity but, if your objective is cloning, I would just keep going and then do some ...
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2 votes
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Optimizing Gel Electrophoresis: Ampere, Volts and Buffer concentrations

As far as I know you can either adjust the ampere or the voltage as both is dependent of the resistance from the buffer. Which means that I would suggest you to let the voltage as it is. As the ...
2 votes

Agarose gel Ladder smear

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 ...
2 votes
Accepted

Why marker and control are loaded in the beginning or ending lane of the gel?

No important reason. It is for esthetics, and also for ease of description in figure legends. One could argue that it might be confusing to have the markers or the control in the middle, but since the ...
  • 7,627
2 votes
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Gel electrophoresis and foam

Gas bubbles are foamed by electrolysis of water, generating bubbles of hydrogen gas on the negative electrode and oxygen gas on the positive electrode. As for the foam itself... I am guessing there ...
  • 2,896
2 votes
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During DNA foot-printing, what is the purpose of radioactively labeling only one end of the DNA fragment?

Preamble Although there is a Wikipedia page with a general account of DNA footprinting, this perhaps does not put sufficient emphasis on the general strategy, making reference to the Maxam & ...
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