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The DNA fragments being run through an electrophoresis gel are being pulled along an electric field. The DNA migrating stays in the gel because the gel is very thick (made from the synthetic polymer polyacrylamide or the seaweed-derived agarose) and the fragments are traveling in a straight line towards the anode (the electrode in which the electricity is flowing into). The wells cut at the beginning end of the gel only go halfway deep into the gel so the traveling DNA is suspended in the middle.
The DNA has an overall negative charge in the sugar-phosphate backbone of the helix, so no matter what the length of the fragment they all move towards the anode when a current runs through the system. Longer pieces lag behind and smaller pieces move quicker through the gel.
Very long DNA segments need to use pulsed-field gel electrophoresis. This method uses and electrical field that is continually making subtle changes in direction. The overall direction stays the same but the sort of snaking motion keeps the DNA oriented in the right direction and from folding over or "catching up with itself".
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Why native gel electrophoresis can be used for determining the molecular weight of proteins?
No! native gels are used to run the proteins in native form, this will tell about the protein's mulimeric nature (ie.monomer or dimer or tetramer etc..).
Why the marker lanes are used during electrophoresis?
The marker lanes are important in electrophoresis because in these lanes peptides or proteins with known molecular sizes and weights (standards) run beside, on the same gel, with the sample and the Rfs (relative mobilities) of the developed bands of the unknown proteins can be compared with those of the standards.
What is the buffer solution and why is it added to the agarose?
A buffer solution is a solution that resists changes in pH when acids or bases are added. It is added to agarose gel to maintain a stable pH environment during electrophoresis, which is critical for optimal separation and visualization of nucleic acids. Agarose gel electrophoresis is commonly performed in a buffer solution such as TAE or TBE.
What is the purpose of gel electrophoresis?
The buffer is the medium through which the current flows. In the electrophoresis chamber, the anode and cathode are separated and the gel is placed between them. In order to close the circuit and generate the voltage which causes the migration, the entire chamber is filled with a conductive buffer. It is actually possible to perform electrophoresis without a buffer; however this requires a specially made electrophoresis chamber. In these chambers the electrodes actually contact the top and bottom of the gel eliminating the need for a conductive buffer to close the circuit.SDS PAGE electrophoresis uses buffer not primarily as a conductor but for holding a desired pH, dissipating heat and providing SDS in excess in the case of denaturing gels. A gel would run without a buffer as the gel itself is a conductor but the currents involved would heat it to the point of decomposition. Also the volume of liquid in a gel does not allow for an adequate pH buffering system. Holding a pH is extremely important for reproducibility especially in native gels as the pH can change the charge on the peptide. It is true some gels do not require buffer but these are rare cases like isoelectric focusing.the primary application of the buffer would be to conduct electricity,to form a closed circuit
How do you avoid smearing of DNA bands in gel electrophoresis?
To avoid smearing of DNA bands in gel electrophoresis, ensure that the gel is properly prepared and poured to have an even surface. Use appropriate voltage and running conditions suitable for the size of DNA fragments being separated. Handle the gel carefully to prevent any unnecessary movement or disruption during loading and running.
