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Questions tagged [gel-electrophoresis]

A method for analyzing or purifying samples. Molecules are separated by their relative weight, charge, or stereochemistry, which affect their speed of movement through a gel.

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39 views

During DNA foot-printing, what is the purpose of radioactively labeling only one end of the DNA fragment?

I read that during DNA foot-printing analysis, DNA is radioactively labeled on one end before being cleaved by DNase 1. I understand that it is labeled so in order to locate the fragment on a gel, but ...
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Protein A-antibody SDS-PAGE

I wanted to know whether the boiling of Protein A- antibody complex for SDS-PAGE analysis will disrupt the interaction? and result in dissociation of protein A and antibody? Thank you
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Number of DNA strands per chromosome

As I was reading Griffith's Introduction to genetic analysis this evidence was provided for single DNA makes single chromosome. Eventually geneticists demonstrated directly that certain chromosomes ...
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How do you interpret the resulting bands of gel zymography?

I would like to clear up some things for gel zymography. I understand that bright bands show proteolytic activity. But which molecular weight do these bands correspond to? Is it the weight of the ...
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Gel electrophoresis and foam

I've never seen this kind of thing happen. What might trigger this??
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Why is the capirally electrophoresis rarely carried out?

I'm a student at the university in Japan. I search details and advantages with each electrophoresis. I think the capirally electrophoresis has much nice method at the points of resolution, rapidity ...
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What is 'exhausted buffer' in gel electrophoresis?

For gel electrophoresis of DNA, we use TAE or TBE. TAE buffer is cheaper than TBE but it has low buffering capacity; it exhausts faster than TBE buffer and there is a need to change the buffer during ...
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31 views

How to confirm secondary structure formation of Precursor miRNA on gel?

I want to fold precursor-miRNA into its secondary structure and then confirm it on gel. At first, I heat it in annealing buffer at 95°C for 5 minutes and cool down slowly. Then I run it on gel but the ...
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56 views

What are the advantages of iTRAQ/TMT technology over electrophoresis?

I'm currently working on the paper regarding proteomics research. I've listed a lot of questions that freshmen may have during their study. Can anyone explain (based on your experience in the lab) the ...
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Why is gel used in electrophoresis?

In analysing amino acids content in a protein through gel electrophoresis, What's the purpose of the gel? Wouldn't putting the amino acid in the gel prohibit the amino acid from dissolving into the ...
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3answers
225 views

What causes DNA gel electrophoresis bands to swirl?

I recently ran a gel on a new system and came out with something I've never seen before. I loaded the first three wells with a ladder in the first and an uncut plasmid in the next two. There are no ...
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How can I differentiate between polysaccharide bands and protein bands on SDS-PAGE? [closed]

I tried to extract bacterial polysaccharide but after running a SDS-PAGE I couldn't differentiate between the polysaccharide band and protein ones. I face a problem of moving up of the samples from ...
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85 views

Which comes first, PCR or Gel electrophoresis?

If I want to find out wither a group of patients have the abnormal BCR-ABL cancer gene, how do I benefit from the PCR and the gel electrophoresis techniques? I'm kinda lost trying to determine which ...
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56 views

Are longer and shorter DNA similarly charged?

A longer DNA molecule would have more phosphate groups, so it should have a greater negative charge, right? It was taught in my class that only terminal ends of DNA are charged and all the phosphates ...
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222 views

Agarose gel electrophoresis - wrong running conditions?

Please have a look at the electrophoregram. The DNA ladder used is GeneRuler 50bp (loaded according to the instructions), size of the gel is 6*7cm, lane width is 4mm, agarose percentage is 1%, TBE ...
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49 views

DNA Sequencing - Sanger termination method: What effects will more ddNTPs in solution have on the resolution on agarose gel via electrophoresis?

Say instead of a small amount of ddNTPs, you add a significant percent of ddNTPs to dNTPs. I was thinking this would reduce the resolution for larger fragments due to more ddNTPs terminating ...
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81 views

Blackgram leaves. SDS page. Phosphate buffer method

I am doing protein analysis in blackgram leaves by using phosphate buffer method. But I cannot get proper bands on my SDS-PAGE. What should I do to get proper bands?
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363 views

Laemmli-SDS-PAGE problems [closed]

I did Laemmli-SDS-PAGE for my Ammonium sulphate precipitate but I had very weak band and have very weird part at the end of gel. Please help me to solve that problem. Thanks
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204 views

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

For forensic inspection, you only obtained red blood cells from an individual and amplified for the D1S80 locus in chromosome 1. Twenty nine different alleles of D1S80 have been identified, and 435 ...
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Why marker and control are loaded in the beginning or ending lane of the gel?

I have seen gel images in several literature. Almost in all, markers and controls are loaded in the extreme lanes (before 1st sample or after last sample) of the gel. I am just curious, is there any ...
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117 views

Gel electrophoresis bands

Is there any way to calculate the number of bands formed due to sodium dodecyl sulphate-polyacrylamide gel electrophoresis for a particular compound of group of proteins? For example, how could I ...
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5answers
597 views

PCR products with no band in gel

I am a PhD student and stuck with the no band in the gel results. I am using the following 1 microliter Primer R (20mM) 1 microliter Primer f (20mM) 1 microliter dNTP (10mM) 1 microliter DNA ...
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2answers
897 views

Agarose gel Ladder smear

We are having trouble with agarose gel electrophoresis. It used to work a couple of months ago but now the ladder always look smeared. We switched the components (1x TBE, 100bp ladder, different type ...
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108 views

Why high quality DNA should be between 10 and 20 kb?

On this website, they are saying that, when using a gel electrophoresis to assess DNA quality, : High quality genomic DNA should give a major band of 10-20 kb on the gel. Why is that so? I ...
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351 views

Quantifying DNA in a band on gel electrophoresis

I am a first year science undergraduate and I am just asking this as a more theoretical question rather than for carrying out a protocol, so I would appreciate if answers do not involve lots of ...
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79 views

What do we call this adjustable platform used to ensure that something is positioned strictly level in a lab?

This is an adjustable platform. Such a platform is used, for example, to make sure that a gel electrophoresis cassette is level relative to the earth. A passage in a procedure description which I'm ...
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2answers
518 views

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

For a lab course we have been assigned to "remove a stop codon, using mutagenesis, that is in the middle of a gene in order for the full gene to be expressed. This will then fluoresce." To do this we ...
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530 views

Why does my gel have such poor resolution?

Brand new user to bio stack exchange here. I ran this gel (0.9% agarose, run starting at 60V then to 105V over ~30-45 min) with DNA samples containing 20uL of DNA and 1X tracking dye in each of the ...
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221 views

Western blot transfer issues

I am having some issues with transfer efficiency to nitrocellulose membranes. To get all the technical info out of the way: Transfer method: Wet, tank transfer overnight at 30V with normal tris-...
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1answer
665 views

Problems with SDS-PAGE, no separation - what could be the problem?

I work in a lab were we have a common stock of 30% Acrylamide, TEMED, TRIS-HCl (pH6.8 and pH 8.8.) and a 10% (w/v) SDS solution pH 7.2. We use the recipe for loading buffer (which we of course mix ...
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2answers
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What are the possible reasons to get extra unrecognized band in agarose gel electrophoresis?

I ran 0.7% agarose gel electrophoresis of my plasmid DNA sample consisting of GFP vector, using NheI and HindIII restriction enzyme. I used two types of plasmids, i.e., isolated by miniprep-alkali ...
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What word/expression is used to describe a band's share in the stained portion of the electrophoretic lane?

I was translating the following sentence from Russian, and realized I was not sure how to put it right: The study shows that the electropherograms of the studied samples of DRUG NAME obtained in ...
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2k views

Why my proteins are migrating like this on SDS-page?

I'm trying to make a SDS-page of total proteins extracted from algae but I'm encountering two problems. First, when I depose my proteins in the wells the sample goes down and then it starts to move up ...
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Transfer of electrophoresis bands to MS

I have a lab question that is confusing me. Just to be clear this is a homework question but I've done some extensive research and can't find what seems to be a "good" answer. The question is this: ...
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1k views

Optimizing Gel Electrophoresis: Ampere, Volts and Buffer concentrations

I am a master student in biochemistry, and I have used gel electrophoresis many times before. What I want to know is how one should adjust the mA (mAmpere) compared to the voltage and the buffer one ...
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Are alleles equally sized?

If alleles occur at the same locus on a chromosome, does that mean they are of the same size? I was under the impression that they were, but I saw a gel electrophoresis testing different alleles of ...
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1answer
3k views

Help analyzing SDS-Page gel

In this experiment, we transformed a truncation of the NFAT protein sequence into a plasmid vector to be expressed in E.Coli as a fusion protein with GST. We also attempted to transform the normal ...
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Why does hemoglobin electrophoresis in sickle cell trait gives two bands but not three?

Electrophoresis of hemoglobin from a patient with sickle cell trait (carrier for sickle cell anemia) shows two bands. Why not three? Since there are two beta chains in each hemoglobin, it seems to me ...
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Which fractions are enriched for siRNA cleavage activity by comparing electrophoresis?

Size exclusion column chromatography was used to separate the proteins in a Drosophila cell lysate to attempt to identify the protein complex responsible for processing long dsRNA into siRNAs. SDS-...
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0answers
1k views

Which DNA fragments do not have expected sizes on this gel electrophoresis?

The problem is such: After performing a PCR, the vector carrying the PCR fragment with two restriction enzymes (Nhe1 and Asc1). The DNA samples were then separated using agrose gel electrophoresis ...
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200 views

Why do I get such a strong background in a detection of DIG-labeled DNA (Southern Blot)?

I have done a Southern Blot Analysis of DNAmt transferred to nylon membranes. The DNA was firstly loaded on a 2% agarose gel. An immunoassay was done to detect the bands using Anti-Digoxigenin ...
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Protocol to dilute DNA step ladder?

I need to run gels that are of not the "most" importance. So I do not want to waste alot of money on step ladder. How do I dilute DNA step ladder? Is there a general procedure, for I have tried ...
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560 views

Freezing after restriction digest

Brief question: I have performed a double digest with heat inactivated restriction enzymes with no star activity. I usually purify the DNA (PCR inserts and linearised vector) using a kit or gel ...
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105 views

Making positive charged polyacrylamide

I am interested in positive charged polyacrylamide to electrophorese molecules I am interested in. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643323/ http://onlinelibrary.wiley.com/doi/10.1002/bip....
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591 views

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

![enter image description here][1] What was possibly wrong in my gel electrophoresis when I didn't see bands of DNA ladder? I'm running a 2% electrophoresis gel (65v for 2h) to confirm the presence ...
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1answer
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Why does my anti-ubiquitin antibody visualization not work on my PAGE gel?

I am using 2D gel electrophoresis to visualize polyubiquitinated proteins. However, while I can see actin and heat shock protein using when appropriate antibodies, I cannot visualize them using anti-...
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1answer
521 views

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

I want to transfect Adipose Derived stem cells using a clone of my own. My problem is that I get a lot of contamination (not only genomic, but due to the nature of the technique, I get undesired ...
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1answer
162 views

Quantitative Trait Locus process?

I do not seem to understand the concept of Quantitative Trait Loci(QTL's), can anyone explain it to me in detail? Reading the wikipedia article helped somewhat, but I do not understand it well. What ...
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Can't resolve protein with native PAGE

This is a native gel. Let's call the left 2 lanes protein A and the right 2 lanes protein B. B is the same as A except it has a FLAG tag. They are both homotetramers of about 65 kDa. After ...
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362 views

Divalent cation binding to calmodulin

I have carried out a native PAGE with 4 reaction mixtures. To each I had added an equal volume of EDTA (1 µl/1mM) to sequester any divalent ions and an equal volume of calmodulin (5 µl/0.5 mg/ml). I ...