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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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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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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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 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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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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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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When running gels what is the difference between constant volts or constant amps?

In general, you want to be consistent with running your gels either at constant volts or constant amps. However, it is very clear that during the progression of both PAGE and agarose gels, the free ...
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What causes skewed lanes in a DNA gel electrophoresis experiment?

In gel electrophoresis, what causes effects like these (see column 11 in the first one, and column 6 in the second)? (These images were samples that I took from an online activity we did for class)...
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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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556 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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DNA extraction from agarose gel

Because DNA is soluble in water, is it possible to extract PCR product by dissolving excised gel containing the DNA band of desired length in water, mashing the gel piece with a pestle, centrifuging ...
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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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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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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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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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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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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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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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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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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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39 views

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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No bands show up in gel electrophoresis, not even marker

I haven't seen any gel lanes in neither my PCR samples nor the marker itself when photographing them. This is not my first time performing 1.2% agarose gel electrophoresis. I see gel lanes when I ...
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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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How do I deal with sticky and viscous samples from cell lysates?

To check a protein expression I pelleted a small amount of E. coli before and after induction and lysed them by redissolving them in SDS-PAGE loading buffer and heating them to 95 °C for 1 minute. ...
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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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106 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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323 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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219 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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497 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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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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478 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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643 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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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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Alternative protocol for evaluating RNA integrity, using a bleach gel

I hate having to run MOPS PFA agarose gels with DEPC and everything just to verify my RNA integrity. I came across an alternative protocol that uses ordinary household bleach in the gel to inhibit ...
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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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Strange behavior of a DNA gel

I ran a PCR product of ~300 bp on a 2% TAE-agarose gel for 30 minutes. I used Sybr-safe as a DNA stain. Voltage was 80V. When I imaged the gel, the DNA on the bottom half of the gel, including the ...
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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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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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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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How do we know that everybody's DNA fingerprint is unique?

How do we know that everybody's DNA fingerprint is unique? I know, I know, everybody's DNA is unique. But when we do DNA fingerprinting, we're looking at very specific regions of high variability. ...
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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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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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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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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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How do I get a brighter DNA bands

I've been trying to a good contrast for my DNA bands, however I haven't been so successful. I've been running the ladders only to make sure I get a good contrast before further experimentation. I ...
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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 ...