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Gel electrophoresis and foam


I've never seen this kind of thing happen. What might trigger this??


Gas bubbles are foamed by electrolysis of water, generating bubbles of hydrogen gas on the negative electrode and oxygen gas on the positive electrode.

As for the foam itself… I am guessing there is some form of detergent in the buffer. Some electrophoresis buffers do contain detergent.


DNA Extraction and Electrophoresis Kits

A complete line of inquiry-based DNA science kits and curricula designed to help teachers introduce students to the exciting world of molecular biology. These educational DNA analysis kits utilize hands-on techniques to explore DNA structure and function, cell structure, restriction digestion, and agarose gel electrophoresis.


Gel electrophoresis and foam - Biology

Principles of DNA Gel electrophoresis

Gel electrophoresis separates DNA fragments by size in a solid support medium (an agarose gel). DNA samples are pipetted into the sample wells, seen as dark slots at the top of the picture. Application of an electric current at the top (anodal, negative) end causes the negatively-charged DNA [remember it's an acid] to migrate (electrophorese) towards the bottom (cathodal, positive) end. The rate of migration is proportional to size: smaller fragments move more quickly, and wind up at the bottom of the gel.

DNA is visualized by including in the gel an intercalating dye, ethidium bromide. DNA fragments take up the dye as they migrate through the gel. Illumination with ultraviolet light causes the intercalated dye to fluoresce with a pale pink colour.

Note that the larger fragments fluoresce more intensely. Although each of the fragments of a single class of molecule are present in equimolar proportions, the smaller fragments include less mass of DNA , take up less dye, and therefore fluoresce less intensely. This is most evident in the lane at the extreme right, which shows a " ladder " set of DNA fragments of known size that can be used to estimate the sizes of the other unknown fragments.


Gel electrophoresis is a common technique used for separation and analysis of DNA, RNA and proteins based on their size and charge. There are two main types of gel electrophoresis namely agarose gel electrophoresis and polyacrylamide gel electrophoresis. Agarose gels are used mainly for nucleic acid separation when higher resolution is required, polyacrylamide gels are used. SDS Page is a type of gel electrophoresis commonly used to separate complex mixtures of proteins. It is considered as a high-resolution protein separation technique. This is the difference between gel electrophoresis and SDS Page.

References:
1. Nowakowski, Andrew B., William J. Wobig, and David H. Petering. “Native SDS-PAGE: High Resolution Electrophoretic Separation of Proteins With Retention of Native Properties Including Bound Metal Ions. www.ncbi.nlm.nih.gov. N.p., May 2014. Web. 7 Apr. 2017
2. Stellwagen, Nancy C. “Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution.” Electrophoresis. U.S. National Library of Medicine, June 2009. Web. 07 Apr. 2017

Image Courtesy:
1. “Gelelektrophoreseapparatur” (CC BY-SA 3.0) via Commons Wikimedia
2. “DNA Agarose gel electrophoresis” By School of Natural Resources from Ann Arbor – DNA lab (CC BY 2.0) via Commons Wikimedia


Gel electrophoresis and foam - Biology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber
[email protected]

Gel electrophoresis is used to characterize one of the most basic properties - molecular mass - of both polynucleotides and polypeptides

  • Gel electrophoresis can also be used to determine
    • the purity of these samples
    • heterogeneity /extent of degradation
    • subunit composition .

    DNA:
    The most common gel electrophoresis materials for DNA molecules is

    The electrophoretic migration rate of DNA through agarose gels is dependent upon four main parameters :

    1. The molecular size of the DNA. Molecules of linear duplex DNA travel through agarose gels at a rate which is inversely proportional to the log of their molecular weight.

    Example: Compare molecular mass vs. expected migration rate:

    1/log (Molec. Mass)
    i.e. relative Mr

    2. The agarose concentration . There is an inverse linear relationship between the logarithm of the electrophoretic mobility and gel concentration.

    inv log(1/Gel %)
    (i.e. relative Mr)

    3. The conformation of the DNA.

    • closed circular DNA ( form-I ) - typically supercoiled
    • nicked circular ( form-II )
    • linear DNA ( form-III )

    These different forms of the same DNA migrate at different rates through an agarose gel.

    • Almost always the linear form (form-III) migrates at the slowest rate of the three forms
    • Supercoiled DNA (form-I) usually migrates the fastest

    Range of separation of linear DNA

    Finally, the DNA being an acidic molecule , migrates towards the positively charged electrode ( cathode ).


    Gel Electrophoresis Exercises

    In this lab, you will use agarose gels to separate DNA molecules produced in PCR reactions. These PCR products should be well-resolved on 1.25% agarose gels prepared in TAE buffer, which provide good separation of molecules that are smaller than 2 kb. Place the casting tray into the gel apparatus. If you are using the BioRad apparatuses, position the black wedges at each end of the casting tray.

    1. Determine the amount of agarose that you will need for a 1.25% (1.25 g/100 mL) gel that fits your casting platform. Most of the gel apparatuses in the lab are the BioRad Mini-Sub GT systems with a 7 cm x 7 cm casting tray that accommodates a 30 mL gel. Check your calculations with your teammates before you proceed.
    2. Fill a graduated cylinder with the appropriate volume of TAE buffer. Pour the solution into a small flask.
    3. Weigh out the appropriate amount of agarose. Sprinkle the agarose onto the surface of the TAE in the flask. Note: the agarose will not dissolve until it is heated.
    4. Dissolve the agarose by heating the solution for intervals of 15-20 seconds in a microwave oven. After each interval, remove the flask and gently swirl it around a bit to disperse the contents. Note if the agarose particles are still apparent or if the agarose has dissolved. The best gels are made from agarose that has NOT been overcooked. SAFETY NOTE: The agarose solution will be very HOT when you remove it from the microwave! Use caution when handling the flask. Be particularly careful not to contact the steam that will be coming through the opening of the flask. Fold several paper towels and wrap them around the neck of the flask when you handle it. If you do happen to spill some hot agarose on your skin, wash it immediately with cold water and alert your TA.
    5. Allow the agarose solution to cool until you can comfortably touch the flask with your hands. Agarose solutions over 60 ̊C will warp the casting tray! Pour the gel. Place the sample comb in place. Do not move the casting platform until the gel sets. You will know that the gel is set when it becomes opaque. Allow the gel to cure for about 20 minutes after it sets.
    Sample preparation

    Prepare your samples for electrophoresis while the gel is curing by adding concentrated loading buffer. The loading buffer contains two dyes, bromophenol blue and xylene cyanol. During electrophoresis, the dyes will migrate with “apparent” molecular weights of

    0.5 kb, respectively. The loading buffer also contains glycerol, which makes the sample dense enough to sink to the bottom of the sample well.

    Add 4 μL of 6X loading buffer directly to each of the 20 μL PCR reactions from the last lab. Briefly, centrifuge each tube to mix the dye and samples, if necessary. You will use half of each sample in your gels. Store the remaining sample in the refrigerator.

    Load and run the agarose gel
    1. When the gel has set, carefully remove the comb and the black wedges.
    2. Orient the gel in the electrophoresis tank such that the wells (holes made by the comb) are oriented toward the black (negative) electrode. DNA molecules will move from the well toward the red (positive) electrode. Fill the tank with enough TAE buffer to submerge the gel (approx. 275-300 mL).
    3. Load one sample to each well, which can accommodate
    Stain and analyze the agarose gel

    SAFETY NOTE: Wear disposable gloves when staining gels. Gloves are important when working with intercalating dyes, which are potential mutagens.


    Spurthi's AP Biology Notebook


    Abstract
    Gel Electrophoresis is a method that separates molecules based on the rate of movement through the gel during the application of an electricity field. The direction the molecules move ( forward or backward) is based on the charge of the molecules because they will move towards their polar opposite. How fast these molecules move is also affected by the physical aspects of the molecule, such as size and shape, the density of the gel, and the strength of the electricity field. Because the density of the gel and the strength of the electricity field is the same for all of the molecules in a given electrophoresis chamber, the smallest particles will have the fastest rate of movement.

    Since we weren’t able to use DNA for this lab, we had to substitute them for loading dyes. These loading dyes contains sucrose, bromophenol blue, and TE buffer. We made the gelatin to have little slots indented on the far end. In those dents, we would fill them up with different color loading dye. The loading dye sunk to the bottom because it contained the sucrose. The Bromophenol blue was the way that they dye was able to move along either being positive of negative. Base on where to dye moved, we can tell how big the molecules in it were and if it was positive or negative.

    Part 1
    First we casted a gel using a waterbath, microwave, 6-well gel comb, masking tap, tray, and agarose gel. Using the masking tape, we covered the two ends of the gel casting tray to block out the agarose gel from falling out. Next we let the gel melt in the waterbath which failed to heat it for long enough. Therefore we microwaved the gel bottle with the cap off in 1 minute intervals until it was completely melted. Using protective gloves we took out the bottle, measured 15 mL of the liquid into a test tube and used the 15 mL in the casting tray. However right before we poured the gel onto the tray, we place the 6-well gel comb near the end of the tray. After allowing the gel to harden on the tray, remove one side’s masking tap. Before carefully sliding the gel without breaking into the chamber, pour buffer from a beaker into one side of the chamber. After placing the gel on the chamber add buffer until the level is approximately 2-3 mm above the top of the gel. Close the chamber and allow it to stay in that position for 24-48 hours.
    Part 2
    2 days later, transfer the gel from the chamber back onto the plate so that it fits perfectly. Next, put the plate with the gel back into the chamber and pour the buffer into the chamber so that it is about 2-3 millimeters above the gel. Using a micropipette, take a color of dye and get 10 ul of it. Repeat this step 4 more times with different colors of dyes and using different micropipettes for each color. Next connect 5 alkaline batteries in a stack and connect the red cord and black cord from the batteries to their respective parts of the chamber. Once the battery pack is connected to the chamber, observe the appearance of bubbles and whether the different colored dyes are moving and if so in which direction of the chamber. After observation, disconnect the battery pack when the fastest moving dye sample is near the end of the gel.


    This was taken within a minutes of activating the electric field. As you can see, most of the dyes have slow rates of movement.

    This photo was taken before disconnecting the wires and stopping the flow of electricity. The results, verified by this photo, are recorded below.

    The length and direction of the run are determined by the charge of the molecule. Based on the results, we can then conclude that the crystal violet dye is the most negative. It is also the only molecule with a negative charge in our lab. Methyl Green was the most positive, followed by the Dye Mixture, Xylene Cyanol, and finally the Bromophenol Blue.

    The crystal violet dye was not in the dye mixture because there was no dye band found in lane 6.

    Certain dyes migrate toward the positive electrode and others toward the negative electrode because in an electrical field, molecules will tend towards to a charge that is the opposite of the one that they carry. Therefore positive molecules will tend towards negative, vice versa.

    Increasing the agarose concentration in a gel will decrease the pore size of the gel once the gel solidified. The smaller pore size may be used to separate a mixture of smaller molecules.

    When the separation of the dyes happened in a higher percentage agarose gel than the ones used to separate many DNA mixtures. We can assume that the dye molecules are generally smaller than the DNA fragments.

    If we were to be called away from our electrophoresis lab and not able to monitor our lab then over time, the dye samples would continue to move towards either the negative or positive side of the poles. If we left it out long enough, then the gel would move off the gel.

    The agarose electrophoresis procedure for DNA is different than our procedure because DNA is not visible to the eye so it requires a tracking dye on the DNA samples to tell when to end the electrophoresis lab. Another difference is that DNA carries a negative charge so the samples are put at one end of the gel instead of the other.

    Electrophoresis Vs. Chromatography

    Electrophoresis Setup


    Another use for electrophoresis could be genetic engineering, protein identification, paternity testing, and screening for genetic disorders. Anything pertaining to DNA can be used in electrophoresis.

    Should data banks be established for DNA information? Data banks should be established for DNA information as long as it is to help others and it is not abused. A benefit of it would be using it to study genetic disorders or identification in court cases but a drawback would be research to try to alter DNA for appearance. People who should have control of the information should be the person’s whose DNA is being stored and information should be controlled by people who are trustworthy. For example, a good person for the job is someone who will not use the DNA bank for his/her benefits. It should also be heavily guarded because it is personal information. DNA taken from a suspect for identification should strictly be used for identification and only identification. Only infants with a family history of a genetic disorder shouldhave their DNA fingerprinted at birth for safety purposes and early prevention purposes.

    Conclusion
    After conducting this experiment using loading dyes, we concluded that the crystal violet dye was the most negative. We also concluded that methyl green was the most positive. This experiment helped us gain a better understanding of gel electrophoresis.


    Trouble Shooting and Cleanup

    Question: "How do you dispose of used petri plates?"

    Answer 1: "Autoclaving is best. If your school cannot afford an autoclave, a good electric pressure cooker (a la Sears) will do the trick. 14# pressure. 15 minutes). You can soak them in 10 percent bleach but that does not kill spores. What if you had anthrax bacilli? These are 'lean, mean, spore-forming machines.' I don't mean to be so lightheaded about it but spore-forming organisms can be as dangerous as toxigenic E. coli."
    Stu Schnell, John C. Freeman High School, Los Angeles, California. 11/30/99

    Answer 2: "Just a suggestion for everyone worrying about used plates. Form a good association with your local hospital. Ours is a designated 'friend of education.' They not only provide us with agar plates and biohazard bags but I return them to the hospital in the biohazard bags and they dispose of them for me along with their waste."
    Marla Vaughn, Oroville, California. 12/6/99

    Answer 3: "I used to take my plates to the microbiology lab at a nearby college. They were kind enough to autoclave them. Some people also pour 10 percent bleach over them, then discard them. However, the laws are changing, and in Connecticut we now have an arrangement with a toxic waste outfit that supplies us with a container lined with plastic. When the container fills up, we call them, and they come pick it up—for a charge (I think we've arranged something like $50 per pickup). It takes a small school like us a long time to fill up the container. As long as you close it thoroughly and contain the smell, it's not so bad. I found this outfit in the yellow pages. We had to make a few calls before we found a company willing to pick up what they call medical waste."
    Barbara Beitch, Hamden Hall Country Day School, Hamden, Connecticut. 12/1/99

    Answer 4: "If you don't have an autoclave or pressure cooker, you can disinfect plates with a 10 percent Clorox solution. Cover and let them sit for 10 minutes."
    Bruce Faitsch, Guilford High School, Guilford, Connecticut. 12/1/99

    Answer 5: "Microwaving is not a safe alternative to autoclaving in my opinion. It doesn't produce enough heat to kill spores, and it often heats the subject unevenly. Use an autoclave or one of the commercial disinfectants that is designed to kill spores. There are professional products on the market that totally disinfect, including killing spores. Wavacide is one such product. The label says that it is a sporicide when used at the recommended strength. I have used it, and it works as far as I can tell. I limit its use to environment, i.e., countertops, floor, etc., if an accidental spill occurs. If you work with bacteria, you should be prepared for spills because they will happen in a classroom, guaranteed! If you are talking about household disinfectants, I agree—they do not kill spores—in fact, they often do not totally kill bacteria vegetative cells. Autoclaving is the preferred method of safe disposal. For teachers not trained in microbiology they probably should use a professional disposal service. I would appreciate any information anyone can provide about a law addressing this issue and, if so, is it a state law, federal law, or just guidelines, because it has been my professional opinion that correctly trained teachers with the proper equipment (autoclave) would be able to safely dispose of bacterial waste. In fact, waiting weeks or even months for a professional disposal is not recommended because the wait may increase the contamination risk."
    Bruce Faitsch, Guilford High School, Guilford, Connecticut. 4/10/00 and 4/11/00

    Question: "Can anyone give me the 'party line' on generating a HindIII standard curve. A few of my students noticed that the EcoRI fragment sizes are much more accurate when the standard curve is generated by just 'connecting the dots' as opposed to constructing a line of best fit. The first data point on the HindIII curve, the largest fragment, is usually way out of line with the other points. So what is it? Is it a 'line of best fit' or 'connect the dots'? On a few university sites, I've read that connecting the points gives more accurate results."

    Answer 1: "When I did this same lab as a student in a graduate molecular biology course, I was told the function was parabolic and to use a best fit curve rather than a best fit line. I have my students in AP Biology do it with the curve and it works great."
    Marcia Sloan, Cleburne High School, Cleburne, Texas. 1/10/01

    Answer 2: "I was taught to drop the top point (the large fragment) in a HindIII curve when using a 1 percent gel because that gel concentration works best for mid-size fragments. To separate larger fragments, a less concentrated gel would be used to separate smaller fragments, a more concentrated gel would be used—thus the various protocols for gel preparation, depending on what size fragments are of the most interest. This gives good results for us. BTW—a few years ago as graphing calculators were being 'pushed' as new technology, students would use those for a best fit curve and got good results. However, since they can't use those on the AP Biology Exam, I also made them use the graph paper. It was amazing to see how many very bright kids couldn't use the graph paper. Now I won't even let them get their calculators out!"
    Ellen Mayo, Mills Godwin Specialty Center for Science, Mathematics, and Technology, Richmond, Virginia. 1/10/01


    What is the difference between Agarose and Polyacrylamide?

    Origin of Agarose and Polyacrylamide:

    Agarose: Agarose is a polymer of natural origin. It is derived from seaweed.

    Polyacrylamide: Polyacrylamide is of synthetic origin and is not found under any natural circumstance.

    Molecular Formula of Agarose and Polyacrylamide:

    Agarose: The molecular formula of agarose is C24H38O19.

    Polyacrylamide: The molecular formula of polyacrylamide is (C 3H5NO)n.

    Chemical Structure of Agarose and Polyacrylamide:

    Agarose: Agarose is a linear polysaccharide. It is made up of repeating disaccharide units called agrobiose held together by hydrogen bonds.

    Polyacrylamide: Polyacrylamide is a chemically cross-linked polymer. It is made up of acrylamide monomers and a crosslinking agent N,N’-methylenebisacrylamide.

    Toxicity of Agarose and Polyacrylamide:

    Agarose: Both agarose and its monomer unit agrobiose are non-toxic in nature.

    Polyacrylamide: The monomer unit of polyacrylamide, the acrylamide, is a presumed carcinogen and known neurotoxin while it’s polymerised form is non-toxic in nature.

    Characteristics of Agarose and Polyacrylamide Gels:

    AGE and PAGE:

    Agarose: Agarose gel preparation for AGE is less time consuming, easy and simple, and does not require an initiator or polymerising catalyst.

    Polyacrylamide: Polyacrylamide gel preparation for PAGE is time-consuming and tedious and also requires an initiator (ammonium persulphate) and polymerising catalyst (N,N,N’,N’-tetramethylethylendiamine – TEMED).

    Nature:

    Polyacrylamide gels are chemically more stable than agarose gels.

    Pore Size:

    Given the same concentration, polyacrylamide gel matrices tend to have smaller pore sizes compared to that of an agarose gel matrix.

    Altering Pore Size:

    The pore size of polyacrylamide gels can be altered in a more controlled manner than that of agarose gels.

    Resolving Power:

    Polyacrylamide gels have high resolving power while agarose gels have low resolving power.

    Accommodating Nucleic Acid:

    Polyacrylamide gels can accommodate larger quantities of nucleic acid than agarose gels for means of resolution.

    Images Courtesy: Agarose and Structure of polyacrylamide via Wikicommons (Public Domain)

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    Watch the video: Gel Electrophoresis (December 2021).