electrophoresis of DNA
Agarose Gel Electroporesis of DNA Making the gel: 1. Place casting platform with well former sideways in gel stand where you wish to pour the gel (preferably in the 4'C cold room). 2. For a 1.2% gel, add 1.2g high purity, wide range Agarose per 100 ml to be made. 3. Add 100 ml of 0.5X TBE (for small gels) or 200 ml of 0.5X TBE (for large gels) into 250ml bottle used and labeled for "DNA gels". 4. Mix by swirling with the cap on, then loosen cap! and microwave for about 2-3 minutes. Stop and mix the solution once or twice during the micro waving. 5. Add 0.5 ug of Ethidium Bromide/ml of gel solution from stock solution (10mg/ml). Thus add 5 ul of stock solution/100ml of gel solution. 6. Pour hot gel into gel cast on a flat surface avoiding bubbles. 7. When gel solidifies, turn gel and tray to proper position and fill gel stand with 0.5X TBE so that it covers gel completely. Loading the Gel: Mix the reaction volumes on a piece of parafilm as follows: 1. First, load appropriate volumes of ddH2O and Loading Dye onto parafilm (see mixture volumes below): 2. One sample at a time, add the appropriate volume of sample and mix with one pipet. 3. Then, with the same pipet tip, load the sample with the loading pipet (one with red tape) into the appropriate well very carefully. 4. Write down what samples are in each well. Mixture volumes (for a total of 12 ul): Standard DNA mixtures: Fragment mixture: Standard DNA 2.0 ul PCR product 5.0 ul ddH2O 8.0 ul ddH2O 5.0 ul Loading dye 2.0 ul Loading dye 2.0 ul Running the gel: Note: DNA and RNA are negatively charges and thus run from positive to negative or from the black to red side if hooked up properly. 1. Add 3-4 ul of Ethidium Bromide stock solution to the bottom of the gel box on the side nearest you (red, positive side). 2. Place lid over apparatus with black lead on the side with the wells. 3. Run gel at 80V for 1-4 hours depending on the size of the DNA (larger DNA runs more slowly than smaller DNA).