-Agarose gels.
Electrophoresis in agarose gels was used to separate, identify, measure and purify DNA fragments. Various concentrations of agarose were used for the gel matrix, depending on the size of the DNA fragments to be resolved. Ethidium bromide was included in the gel itself at a final concentration of Ipg/ml in order to visualize the DNA in UV light. Prior to electrophoresis 1/5 volume of loading buffer (0.2M EDTA pH 8.3, 50% v/v glycerol, 0.05% w/v bromophenol blue) was added to each sample. Electrophoresis was carried out in one of two buffer systems. When DNA fragments were to be recovered from the gel, a TAE buffer was used (40mM Tris/acetate pH 7.7, ImM EDTA). For all other purposes a TBE buffer was used (89mM Tris/borate pH 8.0, ImM EDTA). The samples were electrophoresed at 1 to 10 V/cm o f gel. After
electrophoresis the gel was placed on an ultraviolet transilluminator and the DNA bands were visualized and photographed.
- Acrylamide gels.
Non denaturing acrylamide gels were used to separate products of different mobility in the band shift assay. Glass plates (25 x 20 cm) were thoroughly cleaned with a dilute solution o f detergent, rinsed several times with distilled water followed by ethanol and left to dry. One of the plates was siliconised by spreading about 10 ml of dimethyldichlorosilane solution using a Kleenex paper towel, rinsed twice with distilled water and then with ethanol. The gel was then assembled with 1.5 mm spacers and held in place w ith clips. The gel mix was prepared w ith 8% acrylamide/bisacrylamide (7.73% acrylamide, 0.26% bisacrylamide) in 0.5X TBE. The gel was polymerised with 0.1% (w/v) ammonium persulfate and 0.01% (v/v) TEMED. The gels were run in IXTBE buffer at 4°C. A pre run of 1 hr at 200V was done before loading the samples. The samples were electrophoresed for 2-4 hrs at 200V in a cold room. After electrophoresis the glass plates were cooled down and separated, with the gel remaining attached to the unsiliconised plate. It was then removed from the plate by overlaying it with a sheet of 3MM paper and carefully lifting. The gel was overlaid with “cling film” and dried under vacuum on a gel dryer at 80°C for 30 min. The dried gel was exposed in direct contact with film for 8-24 hrs before the autoradiograph was developed.
2.6.2.- Denaturing gels.
-Sequencing gels.
Glass plates were thoroughly cleaned and rinsed several times with distilled water followed by ethanol and left to dry. The notched plate was siliconised and the gel sandwich assembled, using 0.1mm spacers and clamps. The gel mix (6% acrylamide 8M urea, 0.5X TBE buffer) was primed with 0.1% (w/v) ammonium persulfate and 0.015% (v/v) o f TEMED. The solution was then poured to fill the gel mold and a comb fitted in place. Gels were allowed to polymerize for at least 1 hr before the gel tank was assembled. Once the gel was set, the lower tank was filled with IX TBE and the upper one with 0.5X TBE. The gel was then pre-run at constant power
from left to right. The running conditions were the same as for pre-run. The gel was run until the bottom dye reached the base o f the plate, and a second load of the same samples was done. The gel was then run until the bottom dye o f the second loaded samples reached the bottom o f the glass plate. After electrophoresis the glass plates were cooled down and separated, with the gel remaining attached to the unsiliconised plate. The acrylamide gel was fixed for 15 min with a solution containing 10% methanol and 10% acetic acid. The gel was then removed from the backing plate by overlaying it with a sheet of 3MM paper and carefully lifting. The gel was overlaid with “cling film” and dried under vacuum on a gel dryer at 80°C for 2 hrs. The dried gel was exposed in direct contact with film for 8-24 hrs before the autoradiograph was developed.
-Formaldehyde gels.
Electrophoresis in agarose gels containing formaldehyde was used to separate, identify, measure and purify RNA fragments. As in non-denaturing agarose gels, the concentrations of agarose used for the gel matrix, depend on the size of the RNA fragments to be resolved. The appropriate amount of agarose was melted in water and, once the temperature dropped below 60°C, 5X MOPS running buffer (O.IM MOPS, 40mM sodium acetate, 5mM EDTA) and 37% formaldehyde were added to give final concentrations of IX MOPS buffer and 2.2M formaldehyde. The gels were cast in a chemical hood, and allowed to set for 30 min at room temperature. The samples were prepared by mixing up to 4.5 pi of the RNA sample with 2 pi of 5X MOPS running buffer, 1 pi of ethidium bromide (1 mg/ml), 3.5 pi of formaldehyde (37%) and 10 pi o f formamide. After incubation for 15 min at 65°C, 2 pi o f formaldehyde gel-loading buffer (50% glycerol, ImM EDTA, 0.25% bromophenol blue, 0.25% xylene cyanol) were added to each sample. Immediately before loading the samples, the gel was pre-run for 5 min at 5V/cm in IX MOPS running buffer. The samples were electrophoresed at 3-4 V/cm of gel. After electrophoresis the gel was placed on an ultraviolet transillum inator and the RNA bands visualized and photographed.
One dimensional gel electrophoresis under denaturing conditions (i.e. in the presence o f 0.1% SDS) was used to separate proteins based on molecular size. The gels were assembled in a mini-gel format with 0.75 mm wide Teflon spacers. The separating gel solution (for 15% acrylamide gels) was prepared using 5.63 ml of acrylamide-bis stock solution (37.5% acrylamide, 1% bis-acrylamide), 3.75 ml o f 4X separation gel buffer (1.5M Tris/HCl pH 8.8, 0.4% SDS). 5.62 ml of water, 100 pi of 10% ammonium persulfate, and 20 pi of TEMED. The solution was poured between the glass plates and overlaid with water. Once the separating gel was polymerized, the stacking gel solution was prepared: 0.5 ml of polyacrylamide-bis stock solution, 1.25 ml of 4X stacking gel buffer (0.5M Tris/HCl pH 6.8, 0.4% SDS) 25 pi o f 10% ammonium persulfate and 5 pi o f TEMED, in a final volume o f 4.5 ml. The stacking gel solution was poured on the top o f the separating gel, and an appropriate comb was inserted. When the gel was ready, it was assembled in the gel apparatus and the tank filled with running buffer (5X stock solution: 15.1 g Tris/base, 72 g glycine 5 g SDS, final volume 500 ml). Samples were treated with loading buffer (2X stock: 1.52 g Tris/base, 20 ml glycerol, 2 g SDS, 2 ml 2-mercaptoethanol, 1 mg bromophenol blue, pH 6.8, final volume o f 100 ml), and boiled for 5 min, just before loading in the gel. The gels were run at 20 mA (constant current) until the sample reached the separation gel, and at 30 mA during the separation. After electrophoresis, the gel sandwich was disassembled and the gel transferred to a tray containing fixing solution (10% acetic acid, 20% methanol) for 1 hr. Then the gel was stained with Coomasie blue, and destained with 5% methanol/ 7% acetic acid. Before drying the gel, it was rehydrated with distilled water for 30 min. The gel was then dried at 80°C with vacuum for 1 hr.