Purpose perform electrophoresis using restriction enzymes and lambda DNA understand how a restriction enzyme works analyze a photograph of electrophoresis understand how gel electrophoresis separates DNA molecules in a mixture how to use electrophoresis to separate DNA fragments determine unknown DNA fragment sizes when given DNA fragments of known sizeII. Materials agarose powdercasting tray and combcameracrushed ice containerdistilled waterDNA sampleselectronic scale with tareelectrophoresis box250 ml Erlenmeyer flasksfilm graduated cylinderhoodiceloading dyemicrocentrifugemicropipet and tips1.5 ml reaction tubes and racksrestriction bufferrestriction enzymes (HindIII, EcoRI, BamHI) 10x TEA bufferUV filterUV transilluminator37 C water bathweighing boatIII. ProcedurePlace the weighing boat on the scale and tare. Weigh out 0.8 ml of agarose powder and place it into a 250 ml Erlenmeyer flask.Add 10 ml of 10x TEA buffer and 90 ml of distilled water into a graduated cylinder to create a 1x TEA buffer solution. Add this to the Erlenmeyer flask containing the 0.8 ml of agarose. Dissolve and boil the agarose solution in a microwave, about 2-3 minutes.Place clean bottom of the casting tray in place, and pour in the agarose solution. Place the casting comb in place. Allow gel mold to set undisturbed until almost opaque, about 10 minutes.Fill a graduated cylinder with 50 ml of 10x TEA buffer and 450 ml of distilled water, creating 500 ml of 1x TEA buffer. In each of the four restriction enzyme tubes, combine 1.0 l of restriction buffer, 7.0 l of distilled water, 1.0 l of the specific enzyme (either HindIII, EcoRI, or BamHI). For the control, add no enzyme. Close the caps tightly and place them evenly balanced in the microcentrifuge and spin for 2-3 seconds. Place the tubes in the 37 C water bath. When the gel has solidified remover the comb in a careful straight up motion. Remove the glass plate bottom without disturbing the g...
