Bios 390 Case Study

BIOS 390BIOS 390 LAB 2: Gel Electrophoresis Gel electrophoresis is a laboratory technique used to separate DNA fragments and other macromolecules by size and charge. Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it separates DNA fragments according to size. A standard, or DNA ladder, is typically included so that the size of the fragments in the PCR sample can be determined. The results of a PCR reaction are usually visualized using gel electrophoresis. DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel is stained with a DNA-binding dye. A DNA band contains many copies of the target DNA region, not just a few copies. DNA is microscopic therefore; many copies must be present before it’s visible to the naked eye. This being a huge reason why PCR is an important tool: it produces enough copies of a DNA sequence that we can see or manipulate that region of DNA. Gel electrophoresis involves a gel: a slab of Jello-like material. Gel electrophoresis separates DNA and protein fragments based on size and why one would use agarose gel electrophoresis versus SDS-PAGE. Gels for DNA separation are often made from 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. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds that form pores and is used for larger fragments of DNA, 50 bp or Sodium Dodecyl Sulfate (SDS-PAGE) which form even smaller pores and is used for tiny fragments of DNA and or proteins. SDS is a chemical agent that denatures proteins, disrupting any non-covalent interactions they may have. This makes it, so the charge of proteins is not a factor when they’re separating out onto the gel, and they’re only being separated strictly by size. PAGE or Polyacrylamide Gel Electrophoresis, which is the substance that the gel’s made from. Remember that SDS-PAGE is for small DNA or protein cells (S for small and S for SDS) and agarose is for larger fragments of DNA. At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed. Before the DNA samples are added, the gel must be placed in a gel box. One end of the box is hooked to a positive electrode (anode), while the other end is hooked to a negative electrode (cathode). The main body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct current. To power this you’ll need a certain type of battery to connect everything within the box that contains the gel electrophoresis apparatus with a typical voltage in the range of 80 - 120 V. The buffer fills the gel box to a level where it just barely covers the gel. The end of the gel with the wells is positioned towards the cathode. The end without wells (towards which the DNA fragments will migrate) is positioned towards the anode. Dye will be mixed in with your samples, so you can see them as they’re running which will help you track the movement of the bands later.