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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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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24 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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103 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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51 views

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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52 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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171 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 ...
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54 views

An unknown band appearing in my gel electrophoresis

I am having trouble understanding what is the source of band "2" in the following gel-electrophoresis: In this experiment, we took an E.coli transformant colony and ran its nucleic acids in the ...
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151 views

Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?

Which buffer is best for DNA Electrophoresis and which is best for Protein to be have a sharp bond? Considering a higher electrical conductivity compared to TAE & TBE and the generation of less ...
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79 views

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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79 views

What causes skewed ladder bands on an agarose gel?

I frequently ran into an issue where the bands of my ladder become skewed. What causes this? Here's an example: 0.7% agarose gel, 100V, 60 minutes. Same problem occurs with 1%. I loaded 10 ul of a 20 ...
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117 views

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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372 views

Possible reasons for DNA getting stuck in well

I am in the process of Library preparation for Illumina MI-Seq using mtDNA. Using NEB Hotstart LongAmp Polymerase, I was able to obtain upto 10kb amplicons of mtDNA. However, when I switched to a ...
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31 views

A question on ImageQuantTL

I am confused about using the ImageQuantTL application as a tool to analyse the Urea Gel. My problem is that I am not sure if the 'shape' for calculation should be the same for all samples (like on ...
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152 views

What is the best file format to store gel images?

I store many gel images which are included in my Latex notebooks. The geldoc software normally produces TIFF files, however, Latex only accepts PNG or JPEG. If I am going to store only a single copy ...
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179 views

Single stranded DNA in gel electrophoresis

From http://www.nfstc.org/pdi/Subject05/pdi_s05_m01_01.htm : ... The separation medium contains a denaturant in order that the electrophoresis is conducted on single-stranded DNA fragments. ...
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207 views

Gel Electrophoresis - loading dye and more

I'm a student new to molecular biology coming in from an eco-evo background looking for tips from you molecular pros! It seems that I'm running quite a few gels. The loading dye I'm using is Gel ...
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444 views

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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1k views

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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163 views

What causes skewed lanes in a DNA gel electrophoresis experiment?

In gel electrophoresis, what causes effects like these (see collumn 11 in the first one, an collumn 6 in the second). ? (These images were samples that I took from an online activity we did for ...
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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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457 views

Gel electrophoresis after RNAse treatment

I do not understand how to solve this question. I know that RNAse will cut smaller pieces of RNA. The answer given is A
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2answers
332 views

Gel electrophoresis question

Leena is a molecular biology student. She purifies two fragments of DNA, 800 and 300 base pairs long. These were obtained from a plasmid after digesting it with HindIII. Each of these fragments has a ...
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176 views

Western blotting questions?

everyone. I've just been introduced to the procedure of Western blotting from my reading, though I'm not entirely sure about some points. I'd appreciate it if someone could help me with this. What ...
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890 views

Basic step by step methods for PCR & Gel electrophoresis class

I'm teaching a class how to do PCR & gel electrophoresis soon and would like it if you could check through my basic step by step instructions - it's a while since I've done one. Is there anything ...
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111 views

Should the length of the electrodes in the electrophoresis chamber be proportional to chamber's size?

I am trying to build a small horizontal electrophoresis chamber from scratch. I want to use it for comet assay and I will be using only 1 slide, so it's going to be about 3cm wide, 10cm long and 4cm ...
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121 views

Pump to separate protein/DNA instead of electrophoresis?

I know that DNA molecules or proteins can be separated in electrophoresis because the electrical charge is used to pull the DNA through the gel. So instead of using electrical, can I using physical ...
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637 views

Just how light-sensitive is ethidium bromide?

One lab I was in was paranoid about keeping it in a foil-wrapped conical tube; my current lab leaves it out on the bench (and it works fine for staining gels). It's the same company/concentration in ...
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Separating DNA Fragments by Gel Electrophoresis. Are all the strands for one size the same?

My apologies if my question is too basic, and please point me to a more appropriate forum. I am reading the textbook "Essential Cell Biology" by Alberts et al, and am consulting other sources as ...
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368 views

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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122 views

Effect of doubling volumes of PCR reagents

We ran PCR with a positive control and two lanes of sample. We used the same sample, but in one PCR mixture we used a total volume of 50μL (25μL Taq, 5μL forward&reverse primer each, ...
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339 views

How to do western analysis to lung tissue?

I am working with lungs from mice, and I want to do a western blot analysis to my samples. I am having trouble with this because my images turn out very "messy", with a high background. Perhaps I ...
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224 views

Does GTP-γS (GTP gamma S) bind all GTP-binding proteins?

I've just read an article Rab10 GTPase regulates ER dynamics and morphology - Nature Cell Biology 15, 169–178 (2013) doi:10.1038/ncb2647. In this paper, to identify Rab proteins in ER, first they ...
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352 views

Where does Taq polymerase migrate during electrophoresis?

Assuming a 1% Agarose gel with TAE. Follow up questions: Is there a way to stain for it? Could the polymerase be captured and reused?
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105 views

When running a gel, what could cause a standard to run “faster” than usual?

We are running a gel on the products of a restriction digest to determine the size of an insert. We know the vector backbone size should be ~2.9kb; however, the standard appears to be running too ...
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3k views

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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3k views

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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1answer
197 views

What is the DNA/protein charge ratio?

To study DNA-protein interaction, I want to do a DNA retardation test by mixing the protein with DNA and afterwarts loading it on an agarose gel to see if the DNA migrates slower. I've found some ...
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Is there a free alternative to Gelcompar for comparing banding patterns across multiple gels?

In order to run my microbial community samples from my experiments through DGGE, I was required to use multiple gels. Thus it is necessary to compare banding patterns across more than one gel. ...
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10k views

How many agarose gel bands are typical for circularised DNA

I am aware that circular DNA can be both relaxed and super coiled. However when running an agarose gel of the circular plasmid along with singly digested plasmid with BamHI and HindIII, I see 1 band ...
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1answer
3k views

How do Proteins migrate in MES vs. MOPS

My gels look significantly different in MES (2-(N-morpholino)ethanesulfonic acid) and MOPS (3-(N-morpholino)propanesulfonic acid). That is to be expected. What I don't understand is why the simple ...
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4k views

How do SDS-PAGE gels differ in a Bis-Tris system vs. a Tris-Glycine system?

Protein migrate differently in Bis-Tris and Tris-Glycine gels. I was curious about the actual reasons why. Do certain gel systems result in a tighter resolution than others?
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690 views

Alternatives to TBE buffer for denaturing polyacrylamide gel electrophoresis of DNA and RNA?

I just stumbled upon an article promoting 10 mM sodium borate as an alternative to the well-known TAE and TBE buffers for agarose gel electrophoresis of DNA (Brody & Kern, 2004). They claim that ...
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1answer
4k views

What would cause bands to appear lower on a nonreduced SDS-PAGE gel?

I have a protein gel and I'm comparing the reduced with the non-reduced gel. On the reduced gel, I have a few shorter bands but these bands appear with low intensity. In the non-reduced gel, I'm ...
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448 views

How often can you reuse coomassie stain?

I usually heat to boil in the microwave, and stain for 12 minutes. How often can I reuse this buffer?
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94 views

Is there a cheap and easy way to check for potentially unsafe UV exposure?

My lab has an older large Fotodyne tabletop UV transilluminator for visualizing gels. It has a UV blocking cover that hopefully reduces UV that passes through, but I've always been suspcious as to ...
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5k views

Why should I degas my gel solution for polyacrylamide gels?

In protocols for polyacrylamide gel electrophoresis (PAGE) I often see instructions to degas the gel solution by putting it under vacuum for 10-15 minutes before polymerizing the gel. I usually ...
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2answers
1k views

Alternatives to ethidium bromide for staining small nucleic acids?

While ethidium bromide works well for staining larger single-stranded RNA or double-stranded DNA molecules, it doesn't stain smaller nucleic acids very well. I observed that at around 20 bases and ...