In this advanced experiment, students actually purify the restriction enzyme, EcoRI! This procedure utilizes an ion exchange chromatography step for EcoRI purification. Column fractions are assayed for the enzyme using Lambda DNA and digestion products are identified by agarose gel electrophoresis. Fractions that contain Eco RI are identified and pooled. The total & specific activities are calculated. Recommended for advanced courses.
For 5 purifications.
Time required: 3 hour 15 minutes.
Kit includes: instructions, ion exchange matrix, chromatography columns, E. coli cell extract, equilibration & elution buffer, Lambda DNA, Lambda/Eco RI Marker, KCl, glycerol, dilution & reaction buffers, gel loading solution, agarose, electrophoresis buffer, InstaStain® Ethidium Bromide.
You need: horizontal gel electrophoresis apparatus, power supply, UV visualization system, waterbath, microcentrifuge, microwave or hot plate, UV spectrophotometer & cuvettes, automatic micropipet with tips, ring stands & clamps, 10 ml pipets, lab glassware, ice and ice buckets.