12
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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
5
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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 ...
- 3,667
5
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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 ...
- 8,256
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 ...
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4
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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 ...
- 68k
4
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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 ...
- 51.5k
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
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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
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
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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
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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
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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 ...
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3
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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 (...
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3
votes
Can I column purify instead of gel extract?
Yes, this is perfectly possible. There are a few small caveats though:
do not overload the column with too much DNA, as this will result in lower yields
also do not load an amount of DNA which is too ...
- 51.5k
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 ...
- 51.5k
2
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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 ...
- 17.6k
2
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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 ...
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2
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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, ...
- 679
2
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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 ...
- 679
2
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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 ...
- 161
2
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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 ...
- 7,637
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 ...
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2
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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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2
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PCR and large-scale PCR
I made the large-scale in a falcon tube and then moved the mixture to PCR tubes each containing 50 μL, so the PCR conditions should be the same as the single PCR amplification
If all reagent ...
- 8,256
1
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What causes DNA gel electrophoresis bands to swirl?
It can possibly be one of two problems:
Either the gel poorly prepared, in that it was cooled for too long, resulting in parts that solidified before pouring it into the mold.
This I have seen before,...
- 141
1
vote
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DNA Sequencing - Sanger termination method: What effects will more ddNTPs in solution have on the resolution on agarose gel via electrophoresis?
You are correct. The greater the ratio of ddNTPs to dNTPs, the more frequently ddNTPs will terminate the reaction, resulting in shorter mean fragment size. However, Sanger sequencing is not done with ...
- 7,637
1
vote
Blackgram leaves. SDS page. Phosphate buffer method
You should de-stain your gel longer. I can see at least one thick band at the top, but without de-staining longer you are probably missing other fainter bands.
Also, please explain what you mean by "...
- 715
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PCR products with no band in gel
My first guess would be that you're using too much template DNA for these conditions. Your wells light up quite a bit, 448 ng is also on the high side.
Try lower concentrations of template DNA (1-100 ...
- 1,473
1
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PCR products with no band in gel
I'm not sure about the scale of your ladder but regardless the DNA causing the banding is very small. They are most likely primer dimers. I would check the primer sequences to make sure there isn't a ...
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