Purification of the Restriction Enzyme Eco RI

Edvotek Kit: Purification of the Restriction Enzyme Eco RI

In this experiment, students purify the restriction enzyme, Eco RI.

This procedure utilizes an ion exchange chromatography step for Eco RI 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 and specific activities are calculated.

Recommended for college level courses.

Kit Includes:

• Instructions.
• E. coli RY (Eco Extract (lyophilized); DEAE-Cellulose. 
• Eco RI reaction buffer; 10x equilibration buffer.
• 50% glycerol; KCl; qualified water.
• Lambda DNA; Lambda/Eco RI marker; Eco RI dilution buffer.
• UltraSpec-Agarose™ powder;  concentrated electrophoresis buffer; 10x gel loading solution;  SYBR® Safe stain.
• Chromatography columns.
• 5ml snap-top microcentrifuge tubes; 1.5 mL snap-top microcentrifuge tubes; 50mL conical tube.

You Need:

• Horizontal gel electrophoresis apparatus, power supply, LED transilluminator.
• automatic micropipette with tips,
• balance, water bath, ring stand and clamps, microwave, hot plate or burner
• hot gloves, safety goggles and disposable laboratory gloves, 
• 13 x 100 mm glass test tubes, assorted laboratory glassware,
• permanent markers and tape,
• 1.5 mL microtest tubes, pipette pump,
• 250 mL flasks or beakers,
• distilled or deionized water, 

Some Components Require Freezer Storage

Group Size: For 5 purifications

Time Required:

• Packing column - 45 minutes
• Restriction analysis A - 35 minutes
• Restriction analysis B - 50 minutes
• Gel prep - 30 minutes
• Electrophoresis - 30 minutes
• Staining & destaining - 2 minutes