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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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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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1answer
65 views

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
139 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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1answer
55 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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1answer
39 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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0answers
138 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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30 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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38 views

What causes this shade in gel electrophoresis

Currently in uni I have recently done a exercise of extracting DNA from yeast cell. This is somewhat related to homework, but instead of asking about the answer im wanting to understand why there is a ...
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65 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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1answer
175 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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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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112 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
434 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
614 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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1answer
85 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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267 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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1answer
71 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
357 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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1answer
349 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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1answer
198 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
477 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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0answers
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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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1answer
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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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1answer
42 views

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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1answer
789 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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2answers
2k views

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
2k 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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1answer
1k views

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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0answers
16 views

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
768 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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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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1answer
889 views

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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1answer
495 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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1answer
101 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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530 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
130 views

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
472 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
136 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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0answers
1k views

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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1answer
342 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 ...
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2answers
8k views

How does one calculate how to dilute a solution to working strength? [closed]

If I'm loading a 3.5ul PCR onto an agarose gel, how do I calculate how much of the 6x loading dye to add?
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1answer
339 views

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

I sometimes use denaturing gel electrophoresis on a preparative scale to purify RNA produced by in vitro transcription. The major issue with this is that the sample after extraction from the gel still ...
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3answers
4k views

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

I have come up with what I thought was a clever idea: Store the agarose gels I pour, and only cut as many lanes as I need to run later, minimizing wasted agarose (and wasted effort/time) when I need ...
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1answer
4k views

What is possibly wrong in my gel electrophoresis when I didn't see bands of DNA ladder on gel? [closed]

What was possibly wrong in my gel electrophoresis when I didn't see bands of DNA ladder, and genomic DNA sample on gel ? I could see bands of genomic PCR and RNA solution. Edit Thank you all of guys ...
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How can I get brighter DNA bands?

I amplified my DNA with real time PCR. The amplification curve shows good amplification but not a single band is observed in my gel after gel electrophoresis. What would be the possible reasons? I ...
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2answers
2k views

How do you visualize RNA on a gel?

I have run an in vitro transcription reaction and produced some RNA of a single species and definite size. I would like to visualize it to check if the reaction worked. Can I do this on an agarose ...
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1answer
2k views

Troubleshooting SDS-PAGE of trypsin-treated BSA

I am currently working on SDS-PAGE technique having 20% acrylamide concentration for hydrolyzed BSA protein. Here I attached a gel photo for trypsin hydrolyzed BSA. I don't know , is it a good result ...