Plasmid Analysis: Safety Precautions & Techniques - Prof. James Bougie, Study notes of Biochemistry

Download Plasmid Analysis: Safety Precautions & Techniques - Prof. James Bougie and more Study notes Biochemistry in PDF only on Docsity! Restriction Digestion and Gel Electrophoresis Safety Precautions Goggles, lab coats, and gloves must be worn in this experiment. The ethidium bromide used in the gel electrophoresis part of the experiment is a mutagen; therefore, care must be taken while working with this reagent. Introduction: When a DNA fragment is inserted into a plasmid, it can later be digested out using the same restriction enzymes whose restriction sites are present in the polylinker section of the plasmid. By knowing the map of the plasmid used in a certain experiment and the number of base-pairs in the inserted fragment one is able to tell what fragments are expected to result after digestion. For instance, as shown in figure 1, the pET-23a vector, which was used in the previous experiment, has multiple restriction sites. Cutting with different restriction enzymes will produce fragments of different lengths. Often, one wants to determine the number of nucleotides present in a purified plasmid. Since circular plasmids have secondary structure (supercoiling), they need to be linearized. To linearize a plasmid the best option is to pick an enzyme from the map that cuts only once within the vector and not the inserted gene. Figure 1: Map of pET-23a Vector highlighting different restriction sites In order to visualize the resulting DNA fragments after digestion, the technique of agarose gel electrophoresis is used. In general, macromolecules can be separated based on their size, charge, and other physical properties. DNA fragments are separated by gel electrophoresis based on their size. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose. Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. Circular forms of DNA migrate in agarose differently from linear DNAs of the same mass. Typically, uncut plasmids migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. Purified agarose is found in a powdered form, and is insoluble in water (or buffer) at room temperature but dissolves in boiling water. When it starts to cool, it undergoes polymerization. The sugar polymers cross-link with each other, causing the solution to become a semi-solid matrix. The solidified agarose gel then forms a matrix resembling a very dense spider web through which DNA molecules must travel from the negative pole of the electrode to the positive electrode. Smaller DNA fragments can move through the web easier than the larger fragments therefore ending up more toward the bottom of the gel compared to larger fragments. The following factors affect the mobility of DNA fragments in agarose gels.  Agarose Concentration: Higher concentrations of agarose (restricting the movement of fragments even further) facilitate separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.  Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster than small fragments.  Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used buffers for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength.  Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility. Procedure: Restriction Digestion: 1. Take out 2 sterile microfuge tubes and add 1μL of 10X buffer to each. L of 10X buffer to each. 2. To each of the tubes add 8μL of your plasmid.μL of 10X buffer to each. L of your plasmid. 3. Add 1μL of 10X buffer to each. L of the restriction enzyme, Pst 1, to one of the tubes only (mark as digested sample). Pipet up and down to mix. 4. Place in water bath at 37°C for 1hr.