Exp 9-gel Electrophoresis

GEL ELECTROPHORESIS A. Theory Since the introduction of gel electrophoresis as a means of studying nucleic acids some 10 years ago, this method has become a power and versatile tool in the investigation and characterization of DNA molecules. It is rapid, precise, inexpensive and requires only small amounts of DNA. Gel electrophoresis allows the rapid separation and high resolution of DNA species on the basis of molecular size and conformation. It is therefore an extremely useful method for the estimation of molecular weights of both intact plasmid molecular and linear DNA fragments produced by restriction endonucleolytic cleavage, provided one has standards of known length. With respect to the sizes of the DNA molecules to be separated, either polyacrylamide or agarose is used as the gel material. Only agarose gels will be discussed here since the resolution capacity of this method is sufficient in most cases. Agarose is a polysaccharide extracted from various red algae. When DNA is subjected to agarose gel electrophoresis, it is forced to migrate through the interstices of this network toward the anode (due to its negatively charged phosphate residues) with a migration velocity depending on the followings • Molecular weight of DNA • Conformation of DNA (CCC, OC, Linear) • Pore sizes of the gel (determined by the agarose concentration) • Voltage gradient employed to the gel • Electrophoretic buffer All these parameters interfere with another, but there are some general guidelines, like for example; 1. High voltage gradients are employed for separating small DNA fragments. Better resolution of high molecular weight DNA is archived at low voltage gradient 2. Small pore size (between 0,7 and 1 % agarose) are used for small DNA fragments (up to 107) Large DNA molecules (15-40 x 106) are usually submitted to electrophoresis in low concentration gels (0,3-0,5 % agarose) Agarose gels can be prepared at concentrations ranging from 0,3-2 % (w/v). Either vertical or horizontal slab gels can be used. Horizontal gels are more convenient to handle and more stable, especially at agarose concentration lower then 0,8 %, although it has been reported that vertical gels give sharper bands. Gels are prepared by suspending and boiling the

calculated amount of agarose powder in an electrophoretic buffer until the solutions is completely homogenous and clear. The molten agarose is cooled to 50-60° C before pouring into the tray. After the gel is completely set, samples are loaded after mixing with loading buffer. To increase the density, loading buffer contains glycerol or sucrose (to a final concentration of 5-10 %). Addition of ficoll (1-2 %) avoids the formation of U-shaped bands. A tracking dye may be added as a visible marker; usually bromophenol blue is used at a final concentration of 0,025 %. Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested form them for use). Because they cut within the molecule, they are often called restriction endonucleases. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence 5′GGCC3′ 3′CCGG5′ The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in the circular DNA molecule of the virus phiX174. Thus treatment of this DNA with the enzyme produce 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined. HaeIII and AluI cut straight across the double helix producing “blunt” ends. However, many restriction enzymes cut in an offset fashion. The ends of the cut have an overhanging piece of single stranded DNA. These are called “sticky ends” because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. Any other source of DNA treated with the same enzyme will produce such molecules. Mixed together, these molecules can join with each other by the base pairing between their sticky ends. The union can be made permanent by another enzyme, DNA ligase that forms covalent bonds along the backbone of each strand. The result is a molecule of recombinant DNA (rDNA). Restriction enzyme digested DNA is usually run in Tris-acetate or Tris-borate buffer can be used (largely dependent on plasmid size). Nevertheless, it is necessary to adapt electrophoretic conditions to the specific circumstances of the particular experiment. DNA bands can be visualized by standing with ethidium bromide (EtBr). The sensitivity of this staining technique depends on the concentration of the DNA applied to the gel. The less the amount of DNA, the weaker the fluorescence. Gels can be stained either by including EtBr in the gel (Note: EtBr influences the mobility of DNA to small degree) or after the run by soaking it in EtBr solution (0.5 μl / ml) for 30-60 min. To diminish the background of

fluoresce, it is advisable to destain the gel with redistilled water, if the second way is preferred. DNA bands are than detected by irradiation with UV at 254 nm on a transilluminator. (Note: Irradiation with sort wavelength UV causes damage to the DNA and destroys its function). The illuminated gel is photographed, usually with a Polaroid camera, equipped with a red filter to exclude UV light. DNA fragments may have the same electrophoretic mobility as the marker dye (depending on agarose concentration and voltage gradient). Thus, these DNA bands may be missed since bromophenol blue absorbs fluorescence released from DNA-bound EtBr and obscures the DNA. As mentioned above the mobility of DNA in gels is a function of its molecular weight and within a limited range inversely proportional to its size; the lager size, the slower the rate of migration. Thus agarose gel electrophoresis allows the determination of relative and approximate molecular weight on the basis of electrophoretic mobility in relation to the mobility of DNA standards of known sizes. Mobilities are usually calculated from photograph. Accuracy of the measurement can be improved by photographic enlargement. The distance from the sample to the top of each band is measured (Migration Distance) and the relative mobility is calculated (Migration Distance divided by the length of the gel). A standard curve is constructed by plotting the log of molecular weight of standard against their relative mobilities and drawing a best fit line through these points. In the small size range, linear relationship is obtained. However, migration of very large DNA molecules become more or less independent of size, so that the relationship is no longer linear and a curve is observed at the top of the scale. Molecular weights of the samples are determined by the placing the measured mobilities on the standard curve and interpolating the size. B. Experimental 1. Materials TE (Tris-EDTA) Buffer:

10 mM Trizma Base 1 mM EDTA (pH: 8.0)

Tris-Acetate Buffer:

40 mM Trizma Base 20 mM Acetic Acid 2 mM disodium EDTA (pH: 8.0)

EtBr Solution:

10 mg/ mL

Loading Buffer:

0.25 % Bromophenol blue 0.25 % Xylene cyanol 15 % ficoll

40 % (w/v) sucrose

WARNING: Ethidium Bromide is a powerful mutagen. Use gloves when working with it. 2. Procedure 1. Dissolve 0.4 g of agarose powder in 50 mL 1X Tris-Acetate buffer (0.8 % w/v; gel size ~ 8 x 10 cm) 2. Cool the solution down to 50-60° C and add 4 μL 10 mg/ml EtBr solution 3. Insert the gel into the apparatus and fill the apparatus with 1X Tris-Acetate buffer. 4. Run the gel at 90 V until the tracking dye reaches to the end of the gel (for about 1 hour) 5. After electrophoresis, gel stain the gel is visualized on UV transilluminator (if EtBr is not included in the preparation of the gel, stain the gel with 0.5 mg/mL EtBr solution for 15 min and than destain for 10 min. with deionized water prior to visualization under UV radiation). 1. Draw the bands seen in the gel on the UV transilluminator.

WARNING: The transilluminator emits short wave UV light which will damage skin and eyes, during prolonged exposure and be sure that proper shielding is in place before turning on the transilluminator. 7. Draw the calibration curve and calculate the molecular weight of the unknown fragment.