Agarose gel electrophoresis we will use agarose gel electrophoresis to determine dna fragment sizes, and to quantify dna. When placed in a matrix of agarose and exposed to an electric field, dna molecules will move towards the positive electrode. Smaller molecules move faster than lager molecules because they produce less friction in the agarose matrix.

The distance each dna fragment moves from the gel origin is directly proportional to the size of the dna fragment. If a dna ladder a standard made up of several dna molecules of known length was run on the gel, we can estimate the sizes of the unknown dna molecules. We can also use the dna ladder to estimate the concentration of dna molecules in a given solution. For example, you must decide what buffer to use the concentration of agarose to use how to prepare the dna sample for loading the voltage and run time to use what dna ladder to use see the protocol agarose gel electrophoresis of dna to determine the correct parameters for your application. Learn about how to perform dna gel electrophoresis by reviewing the concepts presented at the following sites:

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Prepare a running buffer with 0.05 g or 1/64 teaspoon sodium chloride, 2.7 g or 1/2 teaspoon sodium bicarbonate, and distilled water, to final volume of 1 l. Place the dried dna snot into about 0.5ml or 1/8 teaspoon of running buffer and let dissolve overnight at room temperature or a slightly warmer location.

After the lab, the tbe buffer solution may be poured back into the container and reused for the restriction digest laboratory. Information for the teacher using a micropipet a micropipet is a very delicate, expensive instrument that is used to dispense an extremely precise and very small volume. It is important to know the volume limits of the micropipet that you are using and to never dial either lower or higher than these limits. The volume settings on the micropipet are generally read from the top to the bottom of the number dials. Often the numbers before the decimal place will be in a different color to those that come after it. For example, on a 200 µl micropipet a volume set at 63.5 µl might have the 6 and the 3 in black lettering but the 5 might be in red lettering.

Using a disposable tip that is the correct size for the micropipet push the shaft of the micropipet firmly down into the sleeve of the tip until a firm seal is made. To aspirate pull in the liquid, first depress the plunger to the first stop, place vertically into the liquid to a depth of about 2 3 mm and slowly release the plunger until it returns to its up position. Now you can move the micropipet out of the liquid and over to the agarose gel well or other vessel. Place the tip against the wall of the well or other surface and begin to slowly depress the plunger, but this time you must move the plunger all the way to the second stop in order to expel all the liquid. While still holding the plunger in the depressed position, move it out of the well or other vessel and then allow the plunger to return to its normal position. If the plunger is released while it is still in the well it will aspirate the liquid that you just deposited there! discard the tip into an appropriate waste container by pressing the ejector button which is found near the top of the micropipet.

Always use a fresh tip for each sample to avoid cross contamination of the samples. buffer solution: tris/borate/edta tbe buffer is commonly used in electrophoresis systems. This salt solution conducts the electric current and controls the ph of the solution during separation of dna fragments, or in this case, dye molecules. Dilute the stock solution as necessary with distilled water to make a 1x solution.

distilled water: minerals in regular tap water will quickly stain equipment. Be careful not to dislodge the wiring at the base of the gel box during this process. gel disposal: when lab is complete, collect all gels in the plastic bags and dispose of in trash. use of power supplies the power supply produces a voltage that is high enough to cause severe electrical shock if handled improperly. Do not plug power supply into wall receptacle until the safety cover is positioned on the cell and all other electrical connections are properly made. For safety reasons, it should not be used with 2 wire receptacle with a conversion plug. Do not operate in a damp or humid environment any condensed moisture may short out electrical components.

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Inspect all power cords, patch cords, banana jacks and plugs for any defects, such as cracked and dried out insulation and loose or wobbly banana jacks or plugs. Do not come in personal contact with or allow metal or any conductive material to come in contact with reservoir buffer or the electrophoretic cell while power supply is on. The power supply may continue to produce some voltage even when the power has been turned off. To eliminate any risk associated with this event, follow all required steps given in the procedure. When disconnecting the gel box, be sure that the leads do not touch each other, come in contact with the buffer solution, or otherwise create any hazardous electrical condition. Agarose is a substance derived from seaweed that forms a jelly like matrix when dissolved in liquid. During electrophoresis an electric current is created through the agarose and molecular fragments can move through the agarose between the two electrodes.

The size of the pores in the gel and the size of the fragment trying to move will determine the rate at which each fragment progresses. The direction in which a fragment moves is determined by the charge that the fragment carries. When using this protocol the granular dyes should be made up in the following concentrations: for all except orange g use 25 mg of dye, add either 10 ml of 60% glycerol solution or 4 g of sugar and make up to 10 ml with distilled water.

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For the liquid dyes, if necessary, dilute with the 60% glycerol solution to the required dilution. Student activity gel electrophoresis of dyes bio molecular laboratory report bio 615 name: nur lisma ruhila bt alias group: as201 5a experiment: gel electrophoresis of extracted dna 0.5% agarose gel group partners: 1 halimatun saadiah bt mohd bustamam 2 nur farhana bt ahmad sopian 3 fatin nur asyiqin bt abd talib. Isbns: 0 471 37972 7 paper 0 471 22390 5 electronic 12 electrophoresis martha l.

electrophoresis and dna fingerprinting jani lynette hagen october 31,2014 u74644799 electrophoresis is a technique which uses an electric field to separate molecules, allowing for identification and characterization of the molecules. Questions: discuss each of the following factors: voltage used. if a higher voltage had been used, the dna would have moved faster through the agar gel, and slower if the voltage was low. Running time. if allowed to run longer, the dna would have eventually ended up into the running buffer, and lost to the experiment. If not allowed to run long enough, the bands could merge and be unclear for reading. Amount of dna. if more dna had been used, the bands would have been darker because more of the fragments would have traveled the same distance in the gel. Reversal of polarity. had the polarity been reversed, the dna would have been drawn the other way through the gel, and ended up in the running buffer.