Concentration of agarose used for separating fragments of different sizes. The drawback of TBE is that the borate ions in the buffer form complexes with the cis-diol groups of sugar monomers and polymers, making it difficult to extract DNA fragments from TBE gels using traditional methods. Gel loading buffer. Pouring the gel Prepare enough 1x electrophoresis buffer both to pour the gel and fill the electrophoresis tank. Add an appropriate amount of agarose depending on the concentration required to an appropriate volume of electrophoresis buffer depending on the type of electrophoresis apparatus being used in a flask or bottle.
Tip: The vessel should not be more than half full. Cover the vessel to minimize evaporation. Tip: Always use the same batch of buffer to prepare the agarose as to run the gel since small differences in ionic strength can affect migration of DNA.
Heat the slurry in a microwave or boiling water bath, swirling the vessel occasionally, until the agarose is dissolved. Tip: Ensure that the lid of the flask is loose to avoid build-up of pressure. Be careful not to let the agarose solution boil over as it becomes super-heated. Tip: If the volume of liquid reduces considerably during heating due to evaporation, make up to the original volume with distilled water.
This will ensure that the agarose concentration is correct and that the gel and the electrophoresis buffer have the same buffer composition. Pour the agarose solution onto the gel tray to a thickness of 3—5 mm. Insert the comb either before or immediately after pouring the gel.
Leave the gel to set 30—40 min. Tip: Ensure that there is enough space between the bottom of the comb and the glass plate 0. Tip: Make sure that there are no air bubbles in the gel or trapped between the wells.
Carefully remove the comb and adhesive tape, if used, from the gel. Fill the tank containing the gel with electrophoresis buffer. Tip: Add enough buffer to cover the gel with a depth of approximately 1 mm liquid above the surface of the gel. If too much buffer is used the electric current will flow through the buffer instead of the gel. Running an agarose gel. Gel loading buffer see table Gel loading buffer must be added to the samples before loading and serves three main purposes: To increase the density of the samples to ensure that they sink into the wells on loading.
To add color to the samples through use of dyes such as bromophenol blue or xylene cyanol, facilitating loading. To allow tracking of the electrophoresis due to co-migration of the dyes with DNA fragments of a specific size. Commonly used DNA markers in agarose gel electrophoresis.
Preparation of samples Add 1 volume of gel loading buffer to 6 volumes DNA sample and mix. Samples should always be mixed with gel loading buffer prior to loading on a gel. Tip: Do not use sample volumes close to the capacity of the wells, as samples may spill over into adjacent wells during loading. Tip: Be sure that all samples have the same buffer composition. High salt concentrations, for example in some restriction buffers, will retard the migration of the DNA fragments.
Ensure that no ethanol is present in the samples, as this will cause samples to float out of the wells on loading. Agarose gel electrophoresis Apply samples in gel loading buffer to the wells of the gel. Prior to sample loading, remove air bubbles from the wells by rinsing them with electrophoresis buffer. Tip: Make sure that the entire gel is submerged in the electrophoresis buffer.
Tip: To load samples, insert the pipet tip deep into the well and expel the liquid slowly. Take care not to break the agarose with the pipet tip. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8.
Determine the sizes of separated DNA fragments. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0.
Add running buffer to the agarose-containing flask. Swirl to mix. This is most commonly done by heating in a microwave, but can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved. Add ethidium bromide EtBr to a concentration of 0. Alternatively, the gel may also be stained after electrophoresis in running buffer containing 0. Note: EtBr is a suspected carcinogen and must be properly disposed of per institution regulations.
Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the staining of DNA are available; however EtBr remains the most popular one due to its sensitivity and cost. Failure to do so will warp the gel tray. Place the gel tray into the casting apparatus. Alternatively, one may also tape the open edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells.
Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box. Add loading dye to the DNA samples to be separated Fig. Gel loading dye is typically made at 6X concentration 0.
Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel. Add enough running buffer to cover the surface of the gel. It is important to use the same running buffer as the one used to prepare the gel. Attach the leads of the gel box to the power supply.
Turn on the power supply and verify that both gel box and power supply are working. Remove the lid. Slowly and carefully load the DNA sample s into the gel Fig. An appropriate DNA size marker should always be loaded along with experimental samples. Replace the lid to the gel box. The cathode black leads should be closer the wells than the anode red leads. Double check that the electrodes are plugged into the correct slots in the power supply. When electrophoresis has completed, turn off the power supply and remove the lid of the gel box.
Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the gel tray on paper towels to absorb any extra running buffer. Remove the gel from the gel tray and expose the gel to uv light. This is most commonly done using a gel documentation system Fig. DNA bands should show up as orange fluorescent bands.
Take a picture of the gel Fig. Figure 5 represents a typical result after agarose gel electrophoresis of PCR products. After separation, the resulting DNA fragments are visible as clearly defined bands.
Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA , RNA and proteins according to their size. Charged molecules move through a gel when an electric current is passed across it. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. The movement of charged molecules is called migration.
Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end opposites attract! The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it.
Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result the molecules are separated by size. DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode.
Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size. The use of dyes, fluorescent tags or radioactive labels enables the DNA on the gel to be seen after they have been separated. They will appear as bands on the gel. A DNA marker with fragments of known lengths is usually run through the gel at the same time as the samples.
How is gel electrophoresis carried out? Preparing the gel Agarose gels are typically used to visualise fragments of DNA. The concentration of agarose used to make the gel depends on the size of the DNA fragments you are working with. The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of DNA are separated on higher concentrations of agarose whilst larger molecules require a lower concentration of agarose.
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