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How to run agar gel electrophoresis at home

How to run agar gel electrophoresis at home


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I've looked at various sites explaining how you can easily make your own gel electrophoresis kit at home. But they always test various food dyes on these home made kits instead of actual DNA samples. Extracting DNA from plant matter can also be done easily at home, so why can't you just run this on the homemade kit?


There are a few reasons why this is usually not suggested.

  • DNA itself is not visible (at least not in the quantities you'd run on your gel). You need a compound (dye) that binds to the DNA to make it visible, and because they bind to DNA most of these compounds are carcinogenic. Even if you use such a dye, many of them only show up under UV light, an additional difficulty. You could also buy a safe dye that works and is visible, but that makes staining also more laborious. Food coloring will be visible all the time, already when running the gel.

  • The nice CSI gels you see on TV are showing (supposed) PCR products of short tandem repeats, giving you nice bands at different heights on the gel. These bands also have a convenient size for gel electrophoresis. Restriction analysis of plasmids is also something you'd see more often, and also here the pieces are between 100 and 10,000 bp. DNA extracted from plants consists of genomic DNA, huge strands (millions of base pairs) that will not really run on an agarose gel. Getting a selection of discrete-sized DNA fragments is really not so easy in your kitchen, that's why they go for food dyes.


Why agarose is used in gel electrophoresis instead of agar?

Agarose gel electrophoresis separates DNA fragments according to their size. An electric current is used to move the DNA molecules across an agarose gel, which is a polysaccharide matrix that functions as a sort of sieve. The matrix helps "catch" the molecules as they are transported by the electric current.

Furthermore, is Agar and agarose the same thing? Difference Between Agar and Agarose. The key difference between agar and agarose is that the agar is a gelatinous substance obtained from red algae while the agarose is a linear polymer purified from agar or red seaweeds. Agar and agarose are two kinds of polysaccharide products that come from red algae or seaweed.

Thereof, can I use agarose instead of agar?

An agarose with low charged groups is therefore generally preferred for use in agarose gel electrophoresis of nucleic acids. As agar is a complex mixture containing Agaropectin, which has sulfate and pyruvate groups, I guess it interacts more with biomolecules than agarose.

What is agarose gel electrophoresis made of?

Gels for DNA separation are often made out of a polysaccharide called agarose, which comes as dry, powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel.


Materials Required:

Agarose solutions.
Ethidium bromide.
Electrophoresis buffer.

Nucleic Acids and Oligonucleotides:

(Samples of DNAs of known size are typically generated by restriction enzyme digestion of a plasmid or bacteriophage DNA of known sequence).
The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:

  • An electrophoresis chamber and power supply.
  • Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.
  • Sample combs, around which molten agarose is poured to form sample wells in the gel.
  • Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
  • Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.
  • Ethidium bromide, a fluorescent dye used for staining nucleic acids.
  • Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.

NOTE: Always wear protective eyewear when observing DNA on a Transilluminator to prevent damage to the eyes from UV light.

For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to 100ml by adding 98ml of distilled water. The 1x working solution is 40 mM Tris-acetate/1 mM EDTA

It is important to use the same batch of electrophoresis buffer in both the electrophoresis tank and the gel preparation.

For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.

Agarose Concentration in Gel (% [w/v])

Range of Separation of Linear DNA Molecules (kb)


Recombinant DNA technology is possible due to several tools useful for manipulating DNA molecules and transforming cells -- including plasmids, restriction enzymes and DNA ligase. This lab introduces you to plasmids and restriction enzymes, as well as the lab technique of gel electrophoresis. Later lab experiments will introduce you to the other tools of biotechnology.

Restriction enzymes (also called restriction endonucleases) are proteins made by many bacterial species, to defend against viral infections. Each restriction enzyme moves along a DNA molecule until it finds a specific recognition sequence in the DNA. The enzyme cuts the double-stranded DNA, resulting in DNA fragments. Over 3000 restriction enzymes that recognize short (4-8 bp) palindromic sequences have been discovered.

Figure 1. Recognition sequence for enzyme Hind III

Figure 1 shows the recognition sequence for restriction enzyme Hind III. Notice that the recognition sequence is a palindrome, and reads the same going forwards and backwards. The Hind III enzyme makes a staggered cut of the DNA, and produces fragments that have single stranded areas called &ldquosticky ends&rdquo. Figure 2 shows the recognition sequence of two other restriction enzymes Sca 1 and Pst 1. Enzyme Pst 1 makes a staggered cut of the DNA at its recognition sequence. But restriction enzyme Sca I makes a blunt cut at its recognition sequence to generate DNA fragments with no sticky ends.

Figure 2: Restriction enzyme recognition sites.

Bacterial cells have all of their genes (genome) in a single circular chromosome. But bacterial cells can also carry non-essential pieces of DNA called plasmids. A plasmid is a small circular DNA that is able to replicate itself, and can carry a few genes from cell to cell. Scientists are able to design recombinant plasmids to carry specific genes into a target host cell.

Figure 3: Plasmid map of pUC19.

The genetic map of a plasmid &ldquopUC19&rdquo is shown in Figure 3. The total size of the plasmid is 2686 bp. There is a Pst I recognition site at position 439, Hind III recognition site at position 447, and Sca I recognition site at 2179. If one restriction enzyme is used to cut pUC19 plasmid, what would be produced?

Determine what DNA fragments are produced when two restriction enzymes are used to cut pUC19 plasmid DNA.

Cut with Restriction enzymes

Sca I and Pst I

Sca I and Hind III

Pst I and Hind III

Resulting DNA fragment sizes


Contents

Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores through which biomolecules can pass. [3] The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state. The melting temperature is different from the gelling temperature, depending on the sources, agarose gel has a gelling temperature of 35–42 °C and a melting temperature of 85–95 °C. Low-melting and low-gelling agaroses made through chemical modifications are also available.

Agarose gel has large pore size and good gel strength, making it suitable as an anticonvection medium for the electrophoresis of DNA and large protein molecules. The pore size of a 1% gel has been estimated from 100 nm to 200–500 nm, [4] [5] and its gel strength allows gels as dilute as 0.15% to form a slab for gel electrophoresis. [6] Low-concentration gels (0.1–0.2%) however are fragile and therefore hard to handle. Agarose gel has lower resolving power than polyacrylamide gel for DNA but has a greater range of separation, and is therefore used for DNA fragments of usually 50–20,000 bp in size. The limit of resolution for standard agarose gel electrophoresis is around 750 kb, but resolution of over 6 Mb is possible with pulsed field gel electrophoresis (PFGE). [7] It can also be used to separate large proteins, and it is the preferred matrix for the gel electrophoresis of particles with effective radii larger than 5–10 nm. A 0.9% agarose gel has pores large enough for the entry of bacteriophage T4. [6]

The agarose polymer contains charged groups, in particular pyruvate and sulphate. [8] These negatively charged groups create a flow of water in the opposite direction to the movement of DNA in a process called electroendosmosis (EEO), and can therefore retard the movement of DNA and cause blurring of bands. Higher concentration gels would have higher electroendosmotic flow. Low EEO agarose is therefore generally preferred for use in agarose gel electrophoresis of nucleic acids, but high EEO agarose may be used for other purposes. The lower sulphate content of low EEO agarose, particularly low-melting point (LMP) agarose, is also beneficial in cases where the DNA extracted from gel is to be used for further manipulation as the presence of contaminating sulphates may affect some subsequent procedures, such as ligation and PCR. Zero EEO agaroses however are undesirable for some applications as they may be made by adding positively charged groups and such groups can affect subsequent enzyme reactions. [9] Electroendosmosis is a reason agarose is used in preference to agar as the agaropectin component in agar contains a significant amount of negatively charged sulphate and carboxyl groups. The removal of agaropectin in agarose substantially reduces the EEO, as well as reducing the non-specific adsorption of biomolecules to the gel matrix. However, for some applications such as the electrophoresis of serum proteins, a high EEO may be desirable, and agaropectin may be added in the gel used. [10]

Factors affecting migration of nucleic acid in gel Edit

A number of factors can affect the migration of nucleic acids: the dimension of the gel pores (gel concentration), size of DNA being electrophoresed, the voltage used, the ionic strength of the buffer, and the concentration of intercalating dye such as ethidium bromide if used during electrophoresis. [11]

Smaller molecules travel faster than larger molecules in gel, and double-stranded DNA moves at a rate that is inversely proportional to the logarithm of the number of base pairs. This relationship however breaks down with very large DNA fragments, and separation of very large DNA fragments requires the use of pulsed field gel electrophoresis (PFGE), which applies alternating current from two different directions and the large DNA fragments are separated as they reorient themselves with the changing current. [12]

For standard agarose gel electrophoresis, larger molecules are resolved better using a low concentration gel while smaller molecules separate better at high concentration gel. High concentrations gel however requires longer run times (sometimes days).

The movement of the DNA may be affected by the conformation of the DNA molecule, for example, supercoiled DNA usually moves faster than relaxed DNA because it is tightly coiled and hence more compact. In a normal plasmid DNA preparation, multiple forms of DNA may be present. [13] Gel electrophoresis of the plasmids would normally show the negatively supercoiled form as the main band, while nicked DNA (open circular form) and the relaxed closed circular form appears as minor bands. The rate at which the various forms move however can change using different electrophoresis conditions, [14] and the mobility of larger circular DNA may be more strongly affected than linear DNA by the pore size of the gel. [15]

Ethidium bromide which intercalates into circular DNA can change the charge, length, as well as the superhelicity of the DNA molecule, therefore its presence in gel during electrophoresis can affect its movement. For example, the positive charge of ethidium bromide can reduce the DNA movement by 15%. [12] Agarose gel electrophoresis can be used to resolve circular DNA with different supercoiling topology. [16]

DNA damage due to increased cross-linking will also reduce electrophoretic DNA migration in a dose-dependent way. [17] [18]

The rate of migration of the DNA is proportional to the voltage applied, i.e. the higher the voltage, the faster the DNA moves. The resolution of large DNA fragments however is lower at high voltage. The mobility of DNA may also change in an unsteady field – in a field that is periodically reversed, the mobility of DNA of a particular size may drop significantly at a particular cycling frequency. [4] This phenomenon can result in band inversion in field inversion gel electrophoresis (FIGE), whereby larger DNA fragments move faster than smaller ones.

Migration anomalies Edit

  • "Smiley" gels - this edge effect is caused when the voltage applied is too high for the gel concentration used. [19]
  • Overloading of DNA - overloading of DNA slows down the migration of DNA fragments.
  • Contamination - presence of impurities, such as salts or proteins can affect the movement of the DNA.

Mechanism of migration and separation Edit

The negative charge of its phosphate backbone moves the DNA towards the positively charged anode during electrophoresis. However, the migration of DNA molecules in solution, in the absence of a gel matrix, is independent of molecular weight during electrophoresis. [4] [20] The gel matrix is therefore responsible for the separation of DNA by size during electrophoresis, and a number of models exist to explain the mechanism of separation of biomolecules in gel matrix. A widely accepted one is the Ogston model which treats the polymer matrix as a sieve. A globular protein or a random coil DNA moves through the interconnected pores, and the movement of larger molecules is more likely to be impeded and slowed down by collisions with the gel matrix, and the molecules of different sizes can therefore be separated in this sieving process. [4]

The Ogston model however breaks down for large molecules whereby the pores are significantly smaller than size of the molecule. For DNA molecules of size greater than 1 kb, a reptation model (or its variants) is most commonly used. This model assumes that the DNA can crawl in a "snake-like" fashion (hence "reptation") through the pores as an elongated molecule. A biased reptation model applies at higher electric field strength, whereby the leading end of the molecule become strongly biased in the forward direction and pulls the rest of the molecule along. [21] Real-time fluorescence microscopy of stained molecules, however, showed more subtle dynamics during electrophoresis, with the DNA showing considerable elasticity as it alternately stretching in the direction of the applied field and then contracting into a ball, or becoming hooked into a U-shape when it gets caught on the polymer fibres. [22] [23]

The details of an agarose gel electrophoresis experiment may vary depending on methods, but most follow a general procedure.


For Next Time

  1. Take the log10 of the length of each molecular weight marker you can identify on your agarose gel photograph. Graph the log10 of their length on the y-axis versus the distance they migrated from the well on the x-axis, measured in mm using a ruler and the picture of your agarose gel. An example of such a graph is found in the introduction to today's experiment. Use the equation of the line from your graph to determine the size of your M13KO7 backbone (use the band in the lane in which you loaded the cut DNA). How does this measurement compare with the predicted size?
  2. How many plaques do you expect if you plated 10 µl of a 10 -8 dilution of phage, if the titer of phage was 10 12 th plaque forming units/ml? How many plaques would you expect if you tested the phage stock on strain DH5?
  3. The oligonucleotide you are adding to p3 uses traditional genetic engineering ("recombinant") techniques. These are powerful and precise ways to move single genes from one organism to another and to make useful chimeric protein products like the one you are now working on. Synthetic biology is a newer approach to programming cells. Please read one (or more!) of the following articles and then write a paragraph exploring the legitimacy of the following statement: "synthetic biology is about engineering while genetic engineering is about biology."
    • Tucker, Jonathan B., and Raymond A. Zilinskas. "The Promise and Perils of Synthetic Biology." The New Atlantis, Spring 2006.
    • Stone, Marcia. "Life Redesigned to Suit the Engineering Crowd." Microbe, Fall 2006.
    • Marguet, P., et al. "Biology by Design: Reduction and Synthesis of Cellular Components and Behaviour." Journal of the Royal Society Interface 4 no. 15 (August 22, 2007): 607-623. (PDF)

White big cloud in agar gel electrophoresis - (Apr/16/2012 )

Hello to everyone. I've been having problem with electrophoresis the past 2 weeks. A white cloud appears in more than upper half of the gel as shown in the picture. I usually make 2% agarose gel with TAE 50x. I make a stock of 1500 mL andI use 50 ml of this solution for the gel, with 3ul of Etrb (10mg/mL). I load the wells with 6ul (5uL dna and 1 uL of dye) For the ladder I use 1 uL of dye and 2 uL of ladder. I dont know what the hell is going on. I decreased the load of Etrb to 3uL ( before I use 5uL), I bought a new dye but the problem persists, also tried changing exposure times in UV transilluminator. I read this could be due to RNA contamination? Because sometimes they use this table to get RNA also. Suggestions will be greatly appreaciated. Thank you very much.

This is called ethidium shadow, and is caused by the ethidium bromide migrating in the opposite direction to the DNA (i.e. towards the -ve electrode). It can be fixed by either adding ethidium bromide to the running buffer or by post-staining your gel.

Note that EtBr is usually used at 0.5 ug/ml not the 20 ug/ml that you are using.

Thank you for your answer Bob, someone in other lab told me the same. I dont understand how adding Etbr to the TAE would make a difference. I usually make the TAE, then the agarose gel, put it 1.30 min in the microwave (take it every 30 seconds and shake it) and after this put the Etbr. Also, I use a 3 ul of a 10 mg/mL EtBr solution do u think im using a bad amount?

Bob was saying to add the EtBr to the buffer in your gel apparatus, in addition to the buffer used to make the gel. You only need it at the positive electrode. The EtBr in the running buffer replaces the EtBr which is migrating in the gel.

Thank you for the advise Phage, im trying this now, will post if i get results or not later, thank you, what about PCR, could inapropiate cycle times in PCR lead to this kind of image later?

This is the image i got today after adding EtBr at the running buffer and after being yelled by my supervisor for thinking this may cause cancer to everyone in the lab even though I covered the glass with tape. I think ill run another PCR

OK. Much better. You are overloading too much DNA on the gel, but that is easy to fix. Your PCR is likely not working. I would say the bands you see are likely primer-dimers, or possibly just primers. Tell us more about your PCR reaction -- template, primers, expected size, cycling conditions, enzymes, everything. What ladder are you using?

Your supervisor thinks adding EtBr in the tank buffer will cause cancer?

Exactly where does he/she think all of the EtBr in the gels migrates to anyway? You could try (gently) pointing out that the tanks are all already contaminated with EtBr and should therefore ALWAYS be handled with appropriate gloves anyway.

Not to mention that the EtBr/cancer hysteria is an overreaction. I'm yet to find any literature on any single case of cancer attributed to EtBr (happy to be corrected. but am doubting there is anything. )


How to run agar gel electrophoresis at home - Biology

Electrophoresis Lab Report:

Calculating Fragment Size of Unknown DNA Molecules

In this lab, a liquid agarose base was used to create a gel base for an electrophoresis procedure using different strands of DNA. Gel electrophoresis is used to separate macromolecules into fragments based on their size. The DNA samples were placed in the wells of the agarose gel at the negative end, and then had a current run through them, causing the DNA to travel a certain length to the positive side through the gel depending on their size. The results were calculated by measuring how far the strands went through the gel, and then mapped on a logarithmic graph.

The purpose of this lab is to explore electrophoresis and DNA manipulation. In the early days of science, DNA was separated by gravity but in the 1950s DNA gel electrophoresis was discovered by Oliver Smithies (Smithies). This process sorts DNA fragments in order of size by using electricity run through a gel matrix (Bowen). DNA is made into different sizes through restrictive enzymes, enzymes that cut DNA into smaller pieces so that they can be analyzed (Pearson). Once the DNA is cut, it is stained then inserted into the wells. When the electricity flows, the DNA is pulled from the negative side to the positive side because DNA has a negative charge (Biology Animation Library). Smaller molecule move more easily while larger molecules move more slowly through the gel. This leads to smaller molecules (and therefore molecule that have been cut by restriction enzymes) travel further toward the negative pole (Biology Animation Library). We are doing this lab to test out this process and see if it successful. Because it has been done so many times by thousands of scientists, we hypothesize that we will be able to separate DNA fragments through DNA gel electrophoresis.

Our electrophoresis lab was conducted on January 28th and 29th of 2016 and was written by Pearson LabBench. We began by using liquid agarose to make a gel base for our DNA molecules to migrate through and placed a comb at the negative end to create wells that would later serve as the site for DNA injection into the gel. Once solidified, we covered the gel in a liquid buffered and allowed the gel tray to refrigerate for 24 hours. Following the period of refrigeration, we removed the comb from the tray to expose a series of formed wells that we could later inject segmented DNA molecules into. We injected the following 5 variations of segmented DNA: Lambda DNA, BamHI, EcoRI, Hindi III and the control (unsegmented DNA.) We then submerged the gel tray with buffer and applied a steady 75V current (Image right) for approximately 15-20 minutes. We then stopped the current, removed the gel and measured and observed any DNA migrations that occurred. Stain was applied to the gel tray to help accentuate any DNA particles present.


Agarose powder, TAE or TBE buffer, Ethidium bromide and bromophenol blue dye.

Gel caster, electrophoresis chamber, voltage source, gel tray, comb, oven, Uv transilluminator.

Pipettes, tips, flask, weight balance.

The hypothetical representation of agarose gel electrophoresis equipment is shown below,

Graphical representation of the electrophoresis apparatus.


RNA analysis on non-denaturing agarose gel electrophoresis

1. The following gel electrophoresis conditions are recommended:

- use 1X TAE buffer instead of 1X TBE - use agarose gel in the concentration of 1.1%-1.2% - add ethidium bromide (EtBr) to the gel and electrophoresis buffer to avoid the additional (potentially RNAse-prone) step of gel staining - always use fresh gel and buffer as well as clean electrophoresis equipment for RNA analysis. Wear gloves to protect RNA samples from degradation by nucleases and avoid a hand contact with EtBr. - use running voltage up to 10 V/cm (10V per each cm of space between the electrodes in electrophoretic chamber). Do not use high voltage to avoid RNA degradation during electrophoresis.

2. Heat an aliquot of the RNA solution at 70°C for 1 min and place it on ice before loading on a gel.

3. Load a known amount of DNA or RNA ladder alongside your RNA sample as a standard for determining the RNA concentration. RNA concentration can be roughly estimated assuming that the efficiency of EtBr incorporation in rRNA is the same as for DNA (the ribosomal RNA may be considered a double-stranded molecule due to its extensive secondary structure).

4. The first sign of RNA degradation on the non-denaturing gel is a slight smear starting from the rRNA bands and extending to the area of shorter fragments. RNA showing this extent of degradation is still good for further procedures. However, if the downward smearing is so pronounced that the rRNA bands do not have a discernible lower edge, this RNA should be discarded.

The following characteristics indicate successful RNA preparation:

- For mammalian total RNA, two intensive bands should be observed against a light smear. These bands represent 28S and 18S rRNA. The ratio of intensities of these bands should be about 1.5-2.5:1. Intact mammalian poly (A)+ RNA appears as a smear sized from 0.1 to 4-7 (or more) kb with faint 28S and 18S rRNA bands.

- In the case of RNA from non-mammalian sources (plants, insects, yeast, amphibians), the normal mRNA smear on the non-denaturing agarose gel may not exceed 2-3 kb. Moreover, the overwhelming majority of invertebrates have 28s rRNA with a so-called "hidden break" (Ishikawa, 1977). In some organisms the interaction between the parts of 28s rRNA is rather weak, so the total RNA preparation exhibits a single 18s-like rRNA band even on a non-denaturing gel. In other species the 28s rRNA is more robust, so it is still visible as a second band.

Note: If your experimental RNA is shorter than expected and/or degraded according to electrophoresis data, prepare fresh RNA after checking the quality of RNA purification reagents. If problems persist, you may need to find another source of tissue/cells. In some cases, partially degraded RNA is only available (e.g. tumor samples or hard treated tissues). This RNA can be used for cDNA preparation, however the cDNA sample will contain reduced number of full-length molecules.

Total RNA from endothelial human cells.

- Commonly, genomic DNA contamination does not exceed the amount seen on the agarose/EtBr gel as a weak band of high molecular weight. Such contamination does not affect cDNA synthesis. DNase treatment to degrade genomic DNA is not recommended. In some cases, excess of genomic DNA can be removed by LiCl precipitation or by phenol:chloroform extraction.